Abstract
Background
Combining chemotherapy with immunotherapy is a promising strategy to
enhance in situ immunity by increasing antigen exposure and
inflammation in the tumor microenvironment (TME). However, the
inconvenience of separate administration and the randomness of delivery
can diminish the synergistic effect of these treatments. An efficient
co-delivery system to optimize chemo-immunotherapy is urgently needed.
Methods
We have previously developed an engineered Lactococcus lactis
(FOLactis) expressing Flt3L and OX40L to activate immune cells. Here,
we biomineralized FOLactis by magnesium (Mg^2+)-based frameworks
(Mg-metal-organic frameworks (MOFs)) loaded with cisplatin and
vincristine (D&V@FOLactis) to achieve sequential activation of in situ
antitumor efficacy. The biomineralized D&V@FOLactis was characterized
by scanning electron microscope. Confocal laser scanning microscopy,
flow cytometry analysis, and immunofluorescence were employed to assess
the biodistribution and the synergistic antitumor mechanisms of
D&V@FOLactis in TME.
Results
On intratumoral administration, D&V@FOLactis exhibited pH-responsive
drug release, primarily inducing immunogenic cell death and enhancing
antigen exposure. Subsequently, FOLactis acted as immunopotentiators to
recruit immune cells and maintain long-lasting effect. D&V@FOLactis was
instrumental in thoroughly improving TME, activating T-cell responses,
suppressing both “cold” and “hot” tumors with abscopal effect, and
prolonging survival. In the clinical trial involving three patients,
the combination of D&V@FOLactis with radiotherapy suggested potential
improvements in systemic immunity and a possible reduction in multiple
types of tumors.
Conclusions
We achieved the sequential activation of synergistic
chemo-immunotherapy using an all-in-one biomineralized strategy. This
study marked the first instance of biomineralizing probiotics with
Mg-MOFs, creating a biosafe and versatile platform for combined in situ
vaccination treatment.
Keywords: Immunotherapy, Chemotherapy, Combination therapy, Vaccine
__________________________________________________________________
WHAT IS ALREADY KNOWN ON THIS TOPIC
* Intratumoral injection of chemotherapeutic drugs combined with
engineered probiotics has shown promise as an in situ cancer
vaccine (ISV)-based chemo-immunotherapy strategy. To fully maximize
the immune responses, the sequential activation of tumor antigen
presentation followed by immunopotentiation is essential. However,
the separate and irregular administration may diminish the
synergistic effect due to the differing pharmacological peaks.
WHAT THIS STUDY ADDS
* We develop biomineralized probiotics D&V@FOLactis for integrating
immunogenic cell death with engineered probiotics. This all-in-one
delivery system sequentially activates the tumor microenvironment
(TME) and systemically recruits diverse immune cells. Additionally,
D&V@FOLactis functions as sensitizing amplifiers enhancing tumor
responsiveness to immunotherapy and thus favoring combination
treatment regimens. In the clinical trial, the combination of
D&V@FOLactis with radiotherapy suppresses tumors and continuously
remodels TME within a short period.
HOW THIS STUDY MIGHT AFFECT RESEARCH, PRACTICE OR POLICY
* This study creates an all-in-one and versatile platform for
combined ISV treatment. It is the first application of
biomineralizing probiotics with magnesium-metal-organic frameworks,
aiming to produce a biocompatible and practical approach for
clinical trials.
Introduction
In situ cancer vaccine (ISV) aims to transform malignant tumors into
immunotherapeutic agents by intratumorally injecting immunomodulatory
agents or through local treatments, overcoming the limitations of
specific antigen identification and delivery.[49]1,[50]3 In a previous
study, we designed an engineered Lactococcus lactis-based ISV
(FOLactis), delivering Flt3L and co-stimulator OX40 ligand (OX40L),
enhancing the maturation and infiltration of dendritic cells (DCs) and
T cells.[51]^4 FOLactis acts as a robust immunologic adjuvant,
requiring the initial exposure of tumor antigens and immune cell
infiltration to elicit immune responses. This highlights the importance
of developing synergistic approaches to directly kill tumor cells and
boost immunogenicity, thereby priming specific immune responses.
Autologous chimeric antigen receptor T-cell (CAR-T) treatments and
neoantigen vaccines have demonstrated efficacy in eliciting specific
immune efficacy, but clinical adoption is hindered by time-consuming
antigen identification and human leukocyte antigen (HLA)
restrictions.[52]^5 6 As a broadly applicable approach,
chemotherapeutic drugs could trigger immunogenic cell death (ICD) in
tumor cells, leading to the release of antigens and numerous
damage-associated molecular patterns (DAMPs), which trigger innate
immune responses.[53]^7 8 Certain drugs, including cis-platinum
(DDP),[54]^9 oxaliplatin,[55]^10 doxorubicin,[56]^11 12 and
paclitaxel,[57]13,[58]15 are investigated for inducing ICD. Notably,
platinum drugs and vinblastine analogs have been proved to effectively
induce ICD and robust cytotoxicity in murine tumor models and clinical
trial when co-formulated at a fixed ratio.[59]^15 Moreover, low-dose
administration of these drugs could elicit ICD-based systemic immunity
while minimizing dose-dependent cytotoxicity. Given the ability to kill
tumors and activate immunogenicity, combining these drugs with FOLactis
is a promising strategy for eliciting specific immune responses.[60]^16
17 However, separate administration and irregular delivery of these
agents lead to inconvenience and uncertain outcomes because of the
varying pharmacological peaks. Therefore, it is necessary to develop an
efficient all-in-one delivery system for probiotic-drug composites to
activate sequentially in tumor microenvironment (TME).
The development of drug-loaded probiotics focuses on genetic
engineering[61]^18 19 and constructing polymeric carriers,[62]^20 21
which are complex processes involving chemical synthesis, tedious
modification, and often limited feasibility. Conversely,
biomineralization offers a convenient alternative for integrating
organisms with metal-organic frameworks (MOFs) to create functional
microbial hybrids.[63]^22 23 MOFs are characterized by excellent
biocompatibility, porosity structure, and functional
modification,[64]^24 making them conducive to loading chemotherapeutic
agents.[65]^25 Accumulating research has been burgeoning in
zinc-MOFs,[66]^26 27 iron-MOFs,[67]^28 29 and manganese-MOFs[68]^30 for
use in biomineralized bacteria, with loading efficiencies that can be
flexibly adjusted to nearly 100% by manipulating the reactant
ratios.[69]^26 31 32 Compared with other metals, magnesium (Mg^2+)
stands out for its broad therapeutic window and safety profile in human
treatments, and is available for intravenous administration. As a
result, Mg-MOFs are an ideal choice for constructing biocompatible and
practical biomineralized probiotics.
In this study, we constructed biomineralized probiotics D&V@FOLactis to
sequentially activate chemo-immunotherapy ([70]online supplemental
scheme 1). Local administration of D&V@FOLactis triggered pH-responsive
drug release, eliminating tumor cells and releasing DAMPs through
inducing ICD. Subsequently, FOLactis expressed Flt3L-OX40L to remodel
TME and elicit potent systemic antitumor immunity. In a clinical trial,
the combination of D&V@FOLactis with radiotherapy (RT) comprehensively
augmented abscopal effect and inhibited tumor growth in patients. This
was the first study of biomineralizing engineered probiotics with
Mg-MOFs, providing a versatile co-delivering platform for combined
ISV-based chemo-immunotherapy.
Results
Synthesis and characterization of D&V@FOLactis
2-methylimidazole (2-MIM) spontaneously conjugates with MgSO[₄] to form
Mg-MOFs for loading DDP and VCR (D&V).[71]^22 By leveraging
biomineralization principles,[72]^33 drug-loaded Mg-MOFs spontaneously
attach to FOLactis through imine bonding and electrostatic
interaction[73]^22 via a one-step in situ method,[74]^26 establishing
D&V@FOLactis ([75]figure 1A). To determine the optimum amounts of
reagents, DDP was used as model drugs, and the concentrations of 2-MIM
and MgSO[4] were adjusted following the ratio of 70:1 to assess
drug-loaded capacity.[76]^26 When the amount of 2-MIM was between 3,500
and 7,000 µmol, the encapsulation efficiency (EE) of DDP remained
nearly unchanged ([77]figure 1B), suggesting loading capacity reached
saturation. However, as the amount of 2-MIM decreased further, the EE
of DDP altered notably ([78]figure 1B). Therefore, the optimum amounts
(2-MIM: 3,500 µmol; MgSO[4]: 50 µmol) were employed to construct
Mg@FOLactis. TEM-element mapping characterization presented Mg^2+
uniformly decorated on the surface ([79]figure 1C), which indicated the
successful biomineralization by Mg-MOFs. And the EE of DDP and VCR was
approximately 40% and 50%, respectively ([80]figure 1D). As shown in
[81]figure 1E, FOLactis exhibited a long and rod-shaped structure with
a smooth surface, while rough biomineralized layers appeared on
D&V@FOLactis, without evident structural changes in morphology
([82]figure 1F). Energy dispersive spectrometer images ([83]figure
1G,H) revealed that characteristic elements, Mg^2+ and Pt^2+, were
distinctly localized on FOLactis. Moreover, the mean diameter of
D&V@FOLactis was increased relative to FOLactis and Mg@FOLactis
([84]figure 1I). Due to the electronegativity of probiotic membranes,
FOLactis exhibited a negatively charged surface property with an
average zeta potential of −27.0 mV ([85]figure 1J). When loaded with
D&V, the average zeta potential increased to −12.2 mV ([86]figure 1J).
Through western blotting analysis, we found enhanced Flt3L-OX40L
protein bands in the lysate sample of D&V@FOLactis,[87]^4 demonstrating
that biomineralized decoration posed negligible impact on the viability
of FOLactis and the production of fusion protein ([88]online
supplemental figure S1).
Figure 1. Synthesis and characterization of D&V@FOLactis. (A) Construction of
D&V@FOLactis and Mg@FOLactis. Created with BioRender.com. (B) Encapsulation
efficiency (EE) of DDP, along with the changes in concentration of
2-methylimidazole (2-MIM) and Mg^2+. (C) Element mapping locating Mg^2+ on
Mg@FOLactis, scale bar: 1 µm. (D) EE, size distribution and zeta potential of
D&V@FOLactis. (E) Scanning electron microscope (SEM) images of FOLactis and
(F) D&V@FOLactis, scale bar: 1 µm. (G) Energy dispersive spectrometer (EDS)
scanning images locating C, N, O, Mg, Pt in D&V@FOLactis, scale bar: 1 µm.
(H) EDS spectrum of SEM-EDS mapping for D&V@FOLactis. (I) Size distribution
and (J) zeta potential of FOLactis, Mg@FOLactis, and D&V@FOLactis (n=3).
CFU,colony-forming unit; DDP, cis-platinum; Mg, magnesium; VCR, vincristine.
[89]Figure 1
[90]Open in a new tab
D&V@FOLactis triggered ICD and immune modulation effect in vitro
The on-demand release of drugs was crucial in killing tumor cells and
inducing ICD. We created acidic environments using tumor cells at
various concentrations to simulate the in vivo TME. 75.43% of DDP
([91]figure 2A) and 58.33% of VCR ([92]figure 2B) was released from
D&V@FOLactis at pH 6.0, whereas only about 2.66% and 2.48% was released
at pH 7.4 within 2 days, confirming the pH-responsive degradation
characteristic of D&V@FOLactis. Additionally, rapid drug release was
observed when mixed with splenocytes (83.2%) ([93]online supplemental
figure S2A) and bone marrow-derived DCs (BMDCs) (49.2%) ([94]online
supplemental figure S2B), due to internalization by immune cells.
Consequently, a CCK-8 assay was conducted to detect tumor-specific
cytotoxicity. Mg@FOLactis showed no significant cytotoxicity to human
umbilical vein endothelial cells (HUVECs) at the experimental doses,
while it exhibited a concentration-dependent cell-killing effect
against 4T1 cells ([95]figure 2C), demonstrating the selective
therapeutic effect of biomineralized carriers against tumor cells. When
encapsulated with D&V, the viability of 4T1 treated with D&V@FOLactis
was significantly decreased compared with other groups ([96]figure 2D).
Figure 2. D&V@FOLactis triggered immunogenic cell death and immune modulation
effect in vitro. (A) Cumulative release of DDP and (B) VCR from D&V@FOLactis
in different pH (n=3). (C) Cytotoxicity of Mg@FOLactis on 4T1 cells and human
umbilical vein endothelial cells (HUVECs) (n=3). (D) Cytotoxicity of
FOLactis, D&V and D&V@FOLactis at various concentrations against 4T1 after
48 hours (n=3). (E) Confocal laser scanning microscopy images of D&V@FOLactis
internalized by bone marrow-derived dendritic cells (BMDCs), scale bar:
10 µm. (F) Flow cytometry analysis and (G) the proportion of matured
dendritic cells (DCs) (CD80^+CD86^+) stimulated by different treatments
(n=3). (H) Representative flow cytometry images and (I) corresponding
quantification of calreticulin (CRT) exposure on tumor tissues after
co-incubation with different treatments (n=3). (J) Representative flow
cytometric analysis and (K) the proportions of matured DCs (CD80^+CD86^+)
co-cultured with 4T1 after stimulation with different treatments (n=3). The
error bars represented mean±SEM. P values were calculated by two-tailed
unpaired Student’s t-tests. ns represented p>0.05. CFU,colony-forming unit;
DDP, cis-platinum; VCR, vincristine.
[97]Figure 2
[98]Open in a new tab
Antigen phagocytosis and presentation by DCs is an essential process
for triggering immune responses. We labeled D&V@FOLactis with DIR and
incubated with BMDCs for 2 hours. Confocal laser scanning microscope
(CLSM) images revealed that D&V@FOLactis was efficiently internalized
by DCs within a short time ([99]figure 2E). To investigate the
activation of DCs, different formulations were incubated with BMDCs for
48 hours. D&V@FOLactis induced the highest proportion of matured DCs at
50.3% ([100]figure 2F,G). Given that DNA damage could self-activate
antigen-presenting cells (APCs) and release pro-inflammatory
cytokines,[101]^9 D&V@FOLactis-treated BMDCs exhibited the highest
level of cytokines secretion, encompassing interleukin (IL)-4,
interferon (IFN)-γ and tumor necrosis factor-α ([102]online
supplemental figure S3). Surprisingly, there was no significant
toxicity to BMDCs at the same concentrations applied in activation
experiments ([103]online supplemental figure S4). We then detected the
representative biomarker calreticulin (CRT) to assess the ICD.
D&V@FOLactis generated the most robust CRT exposure, which was 2.10
times higher than that of free chemotherapeutic drugs ([104]figure
2H,I). This was attributed to the sustained-release and nanoscale
properties of Mg-MOFs that facilitated internalization by tumor cells,
allowing for effective action on intracellular targets and highlighting
the crucial role of the biomineralized co-delivery system. Since DAMPs
are stimulation signals for APCs, all formulations were co-incubated
with BMDCs and 4T1 to simulate the TME. And D&V@FOLactis exhibited the
highest maturation rate of DCs ([105]figure 2J,K). Notably, the
proportion of matured DCs in D+V+FOLactis groups was lower than that in
D&V@FOLactis groups, probably because the co-delivering system confined
the ICD and FOLactis to concentrated areas, resulting in superior
performance and implying the biomineralized co-delivery system, rather
than simply mixing D&V with FOLactis, was essential for initiating
immune responses.
D&V@FOLactis induced potent antitumor effect in vivo
We first monitored the biodistribution of D&V@FOLactis by labeling with
DIR. The images revealed that D&V@FOLactis predominantly accumulated at
tumor sites ([106]figure 3A,B and [107]online supplemental figure S5A)
and migrated to tumor-draining lymph nodes (TDLNs) within 24 hours
post-administration ([108]figure 3B and [109]online supplemental figure
S5B). During the initial week, D&V@FOLactis showed higher accumulation
of viable bacteria in tumors and TDLNs than FOLactis ([110]figure
3C,D). And D&V@FOLactis maintained colonization for over 2 weeks. There
were no detectable signals in other organs except for the presence in
livers and spleens due to metabolism ([111]figure 3E), suggesting the
biosafety of D&V@FOLactis.
Figure 3. D&V@FOLactis induced potent antitumor effect in vivo. (A) In vivo
biodistribution images of 4T1 tumor-bearing mice following intratumoral
administration of FOLactis and D&V@FOLactis, and (B) ex vivo biodistribution
images of mice treated with D&V@FOLactis at different time intervals (n=3).
(T) Tumors, (L) TDLNs. (C) The comparison of bacterial colonization in tumors
and (D) TDLNs at different time points following FOLactis and D&V@FOLactis
treatment (n=3). (E) Corresponding fluorescence intensities of various organs
harvested from 4T1-bearing mice at different time intervals after injection
of D&V@FOLactis (n=3). (F) Schematic illustration of the therapeutic
procedures. Created with BioRender.com. (G) Tumor growth curves of CT26
tumor-bearing mice after intratumoral administration with different
formulations (n=6). (H) Survival and (I) body weight changes in CT26
tumor-bearing mice during the therapeutic process (n=6). (J) Tumor growth
curves and (K) corresponding tumor weights of 4T1-bearing mice after
treatments with different formulations (n=6). (L) Body weight changes of
4T1-bearing mice during the therapeutic process (n=6). (M) Tumor photographs
and (N) individual tumor growth curves of sacrificed 4T1-bearing mice
following treatment with different formulations (n=6). The error bars
represented mean±SEM. P values were calculated by two-way analysis of
variance (G, J) or log-rank (Mantel-Cox) test (H). ns represented p>0.05.
CFU,colony-forming unit; i.t, intratumoral injection; TDLNs, tumor-draining
lymph nodes.
[112]Figure 3
[113]Open in a new tab
Next, we assessed the antitumor effect of D&V@FOLactis in vivo. First,
we established CT26 tumor-bearing mice and intratumorally administered
different treatments following a four-time injection regime
([114]figure 3F). As depicted in [115]figure 3G D&V@FOLactis eliminated
tumors with an 88% tumor inhibition rate, which was 7.3-fold and
1.3-fold higher than that of FOLactis (12%) and D&V (67%),
respectively, and demonstrated the superior synergistic effect between
ICD and probiotics. All tumors treated with D&V@FOLactis were below
200 mm^3, and even 1/6 of the mice achieved complete regression (CR)
without tumor recurrence. Furthermore, D&V@FOLactis significantly
prolonged survival time, with one mouse confirmed to achieve long-term
survival over a 6-month observation period ([116]figure 3H).
D&V@FOLactis did not cause obvious changes in body weights ([117]figure
3I).
Given that CT26 tumor models exhibited sensitivity to immunotherapy, we
next evaluated the broad applicability of D&V@FOLactis in 4T1 mouse
model, which had minimal immune cell infiltration ([118]figure 3F). As
shown in [119]figure 3J,K, the tumors in D&V@FOLactis were
significantly smaller, with an inhibition rate of 74.8%. This
represented a 2.1-fold and 1.8-fold increase in efficacy compared with
FOLactis and D&V, respectively. Throughout the treatment, there was
only a slight fluctuation in body weights ([120]figure 3L).
Post-treatment, the tumors were excised for photographing ([121]figure
3M), and the results were consistent with the tumor growth curves
([122]figure 3N). Furthermore, Mg@FOLactis showed negligible tumor
inhibition efficacy, suggesting that the immune effect of probiotics
combined with metals was limited ([123]online supplemental figure S6).
The H&E staining of major organs revealed no noticeable damage in any
groups ([124]online supplemental figure S7), and serum biochemistry
indices remained within a normal range ([125]online supplemental figure
S8A,B), indicating the biocompatibility of D&V@FOLactis.
D&V@FOLactis triggered ICD and potentiated chemo-immunotherapy
To elucidate the antitumor mechanisms of D&V@FOLactis, we assessed
various immune cell populations in tumors, TDLNs, and spleens. We
repeated the treatment as previously described for 4T1 tumor-bearing
mice ([126]figure 3F) and sacrificed 7 days post the final dosing. In
general, D&V primarily exerted antitumor effects through ICD, and when
combined with FOLactis via biomineralization, the exposure of CRT was
particularly pronounced in D&V@FOLactis (57.0%), which was 1.86-fold
higher than that observed with FOLactis (30.5%) ([127]figure 4A,B).
Mechanically, D&V@FOLactis had the potential to combine ICD with
Flt3L-secreting FOLactis, facilitating the maturation of DCs. As
anticipated, D&V@FOLactis increased the infiltration of matured DCs to
1.45%, compared with 1.10% and 0.98% in mice treated with FOLactis and
D&V, respectively ([128]figure 4C,D). DDP[129]^34 and Mg^2+ [130]^35 36
have been reported to modulate immune responses in T cells, and with
OX40L-expressing FOLactis, the proportions of CD4^+ and CD8^+T cells
were upregulated in D&V@FOLactis (33.3% and 24.9%) ([131]figure 4E–G).
Strikingly, in the D&V@FOLactis group, CRT exposure predominantly
occurred around the tumor periphery, disrupting the structure and
creating avenues for immune cell infiltration into the tumor core,
whereas in other groups, most immune cells were halted at the tumor
edges due to the dense fibrotic stroma in solid tumors, leading to
insufficient infiltration ([132]figure 4H). Given the immune regulation
effect of Mg^2+ on CD8^+ T cells,[133]^35 a substantial accumulation of
cytotoxic T cells was observed in the tumor center of
D&V@FOLactis-treated mice compared with those treated with D+V+FOLactis
([134]online supplemental figure S9). Additionally, an apparent
downregulation of regulatory T cells was observed in the D&V@FOLactis
group, which was about 1/5 and 1/2 of those in the FOLactis and D&V
groups, respectively ([135]figure 4I). The highest M1/M2 ratio was also
found in the D&V@FOLactis group, indicating an enhanced ability to
remodel TME ([136]figure 4J). Consistently, D&V@FOLactis induced a
significant proliferation of cytotoxic T cells in both TDLNs
([137]figure 4K,L) and spleens ([138]figure 4M,N). In conclusion, the
co-delivery of chemotherapeutic drugs and engineered probiotics held
promise for eliciting robust immune responses by translating ICD
effects into immunotherapeutic efficacy.
Figure 4. D&V@FOLactis triggered immunogenic cell death and potentiated
chemo-immunotherapy. (A, B) Flow cytometry analysis of CRT expression and (C,
D) the frequency of matured dendritic cells (CD80^+CD86^+) in 4T1 tumor
tissues following various treatments (n=5). (E) Representative
immunofluorescence images of 4T1 tumor cells after injection with NS and
D&V@FOLactis (scale bar: 50 µm), and the proportions of (F) CD3^+CD4^+ T
cells and (G) CD3^+CD8^+T cells assessed by flow cytometry (n=5). (H)
Immunofluorescence images of 4T1 tumor tissues in different treatment groups.
(I) The proportions of regulatory T cells (CD3^+CD4^+/CD25^+Foxp3^+) and (J)
the ratio of M1-like to M2-like macrophages infiltrating in the tumor tissues
(n=5). (K) The proportions of CD3^+CD4^+ T cells and (L) CD3^+CD8^+ T cells
in TDLNs (n=5). (M) Representative flow cytometry images and (N)
corresponding proportions of CD3^+CD8^+ T cells in spleens (n=5). The error
bars represented mean±SEM. P values were calculated by two-tailed unpaired
Student’s t-tests. ns represented p>0.05. CRT, calreticulin; TDLNs,
tumor-draining lymph nodes.
[139]Figure 4
[140]Open in a new tab
Chronological changes in TME stimulated by D&V@FOLactis
To thoroughly elucidate the synergistic mechanisms between ICD and
probiotic-induced immunity, we investigated the temporal dynamics of
TME in 4T1-bearing mice following a single intratumoral injection
([141]figure 5A). D&V@FOLactis (51.4%) induced the highest level of CRT
expression and was closely followed by the D&V group (45.8%) within
72 hours, suggesting that ICD was predominantly mediated by drugs
acting as immune activators ([142]figure 5B). Importantly, tumors
treated with D&V@FOLactis sustained high levels of CRT expression for
up to 7 days ([143]figure 5C). Concurrently, we observed that at
72 hours post-treatment, the D&V@FOLactis group exhibited a significant
increase in the percentages of DCs ([144]figure 5D,E) and CD8^+IFN-γ^+
T cells ([145]figure 5F,G) at the tumor sites, with 17.5% and 10.5%,
respectively, compared with FOLactis (12.5% and 6.1%) and D&V groups
(11.9% and 2.9%). Due to the long-term immune responses induced by
probiotics, mice treated with both FOLactis and D&V@FOLactis maintained
high levels of DCs at 96 hours and stably recruited CD8^+IFN-γ^+ T
cells over 7 days, which rapidly decreased in the D&V group after
96 hours. These findings suggested that drugs can attack tumor cells
and rapidly activate immune responses, with FOLactis sustaining the
prolonged immune response. Additionally, the highest proportion of
matured DCs was significantly infiltrated in TDLNs at 72 hours
post-administration with D&V@FOLactis (10.8%) ([146]figure 5H,I),
indicating the capacity to evoke systemic immune efficacy as a
promising ISV. Overall, consistent with its in vivo distribution,
D&V@FOLactis achieved optimal therapeutic effect within 72 hours,
facilitated robust antitumor immune responses through sequential
activation of chemo-immunotherapy.
Figure 5. Chronological changes in TME stimulated by D&V@FOLactis. (A)
Schematic illustrations of the therapeutic procedures. Created with
BioRender.com. (B) The proportions of CRT exposure, (D) matured DCs
(CD80^+CD86^+) and (F) CD8^+IFN-γ^+ T cells in tumor sites, as well as (H)
matured DCs (CD80^+CD86^+) in TDLNs, were assessed in 4T1-bearing mice at
48 hours, 72 hours, 96 hours, and 168 hours post-treatment with different
formulations (n=3). (C) The corresponding change curves for CRT exposure, (E)
matured DCs (CD80^+CD86^+) and (G) CD8^+IFN-γ^+ T cells in tumor sites, as
well as (I) matured DCs (CD80^+CD86^+) in TDLNs, within 168 hours after
intratumoral injection with different treatments (n=3). The error bars
represented mean±SEM. P values were calculated by two-tailed unpaired
Student’s t-tests. ns represented p>0.05. CRT, calreticulin; DCs, dendritic
cells; IFN, interferon; TDLNs, tumor-draining lymph nodes.
[147]Figure 5
[148]Open in a new tab
D&V@FOLactis inhibited the growth of distant tumors with an abscopal effect
To assess the abscopal effect, we established bilateral 4T1 tumors to
simulate metastatic tumors, applying treatments exclusively to the
primary tumors ([149]figure 6A). D&V@FOLactis remarkably reduced
primary tumor growth, achieving an 84.3% tumor inhibition rate
([150]figure 6B), which was 2.1-fold and 1.6-fold higher than that of
FOLactis (41.0%) and D&V (50.9%), respectively. In the D&V@FOLactis
group, all tumors remained below 250 mm³ and 2/6 tumors achieved CR.
Although D&V@FOLactis did not exhibit evident tumor suppression effects
in distant tumors ([151]figure 6C,D), it slowed tumor growth and
prolonged survival compared with other groups ([152]figure 6E),
illustrating the combined application of ICD and engineered probiotics
suppressed tumor metastasis to some extent. Furthermore, the tumor
volumes in the D&V@FOLactis group were remarkably smaller than those in
the D+V+FOLactis group ([153]online supplemental figure S10). This was
attributed to the biomineralized delivery system, which could prolong
the retention at tumor sites and enable D&V and FOLactis to act on the
same targets compared with free components. In essence, D&V@FOLactis
achieved spatiotemporal control over immune responses.
Figure 6. D&V@FOLactis inhibited the growth of distant tumors with an
abscopal effect. (A) Schematic illustration of therapeutic procedures.
Created with BioRender.com. (B) Tumor growth curves for primary tumors and
(C) distant tumors in 4T1 tumor-bearing mice following various treatments
(n=6). (D) Tumor photos of sacrificed 4T1-bearing mice treated with NS and
D&V@FOLactis on D20 post-treatment (n=4). (E) Survival of 4T1-bearing mice
throughout the treatment period (n=6). (F) Representative flow cytometry
images and (G) the corresponding proportions of CD8^+IFN-γ^+ T cells in
primary and distant tumors (n=5). (H) Representative flow cytometry images
and (I) the proportions of effector memory T cells (CD44^+CD62L^−/CD3^+CD8^+)
in primary and distant tumors post-treatment (n=5). (J) The proportions of
matured dendritic cells (CD80^+CD86^+), (K) CD3^+CD4^+ T cells and (L)
CD3^+CD8^+ T cells in TDLNs (n=5). The error bars represented mean±SEM. P
values were calculated by two-way analysis of variance (B, C), log-rank
(Mantel-Cox) test (E) or two-tailed unpaired Student’s t-tests (G, I, J–L).
ns represented p>0.05. IFN, interferon; i.t, intratumoral injection; TDLNs,
tumor-draining lymph nodes.
[154]Figure 6
[155]Open in a new tab
We sacrificed mice from all groups and collected tumors and TDLNs for
flow cytometry analysis. IFN-γ was reported to broadly modulate
antigen-negative cells in TME and was essential in initiating distant
immune responses.[156]^37 D&V@FOLactis stimulated the highest
percentage of CD8^+IFN-γ^+ T cells (2.59% and 13.28%) in both primary
and distant tumors ([157]figure 6F,G). Additionally, an apparent
increase of effector memory T cells was found in D&V@FOLactis (12.48%
and 12.92%), which renders the capability in promptly initiating immune
responses to tumor recurrence and metastasis ([158]figure 6H,I). The
infiltration of immune cells into TDLNs is a key determinant in
eliciting systemic immune responses. D&V@FOLactis induced a dramatic
increase in matured DCs ([159]figure 6J) and T cells ([160]figure 6K,L)
in TDLNs, demonstrating the robust abscopal effect of D&V@FOLactis.
D&V@FOLactis activated innate and adaptive immunity
We compared the differences in transcriptional characteristics between
NS and D&V@FOLactis samples using RNA-sequencing (RNA-seq) data
analysis. We found that 1,066 differentially expressed genes (DEGs)
were upregulated and 469 DEGs were downregulated ([161]online
supplemental figure S11). Genes involved in immune cell regulation and
antigen presentation were significantly upregulated in D&V@FOLactis
samples ([162]figure 7A), including Il6, Ccl3, Cxcl10, Cxcl5, etc
([163]figure 7B). Subsequently, the Kyoto Encyclopedia of Genes and
Genomes (KEGG) ([164]figure 7C) and Gene Ontology (GO) analyses
([165]figure 7D) illustrated that D&V@FOLactis influenced multiple
immune pathways, such as cytokine activity, lymphocyte chemotaxis, and
cell killing. In particular, pathways related to metal ion
transmembrane transporter activity were enriched in D&V@FOLactis
relative to NS group, emphasizing the efficient transfer of Mg^2+ and
Pt^2+ into tumor cells and APCs to enhance STING activation.
Concurrently, Mg^2+ and FOLactis co-regulated the activity of cGAS and
toll-like receptors (TLRs) to further enhance APC activation.[166]^38
As such, DEGs analysis showed the genes related to the ICD, DNA-sensing
pathway, TLR signaling pathway, and cyclic GMP-AMP synthase (cGAS)-
stimulator of interferon genes (STING) signaling pathways were
upregulated by D&V@FOLactis ([167]figure 7E). In summary, D&V@FOLactis
potentiated innate and adaptive immune responses via
chemotherapy-induced ICD, engineered probiotics, and
metalloimmunotherapy, acting in the cGAS-STING pathway and TLR pathway.
Figure 7. D&V@FOLactis activated immune responses and improved clinical
outcomes in patients. (A) Heatmap of differentially expressed genes (DEGs) in
various immune cells between mice treated with NS and D&V@FOLactis (n=3). (B)
The protein–protein interaction network of significant upregulated
immune-related genes in D&V@FOLactis samples compared with NS samples. Color
scales and circle size represented the degree of the interaction of genes
(n=3). (C) Kyoto Encyclopedia of Genes and Genomes (KEGG) and (D) Gene
Ontology (GO) pathway enrichment analysis of DEGs in D&V@FOLactis group. (E)
Heatmap of selected DEGs related to the ICD effect, cytosolic DNA-sensing
pathway, cGAS-STING signaling pathway, and toll-like receptor signaling
pathway (n=3). (F, I, L) Schematic illustrations of the therapeutic
procedures for various patients. Created with BioRender.com. (G) Comparison
of perineal tumors and (H) inguinal lymph node tumors before and after
treatment in patient 1 using MRI and CT scans, respectively. (J) Comparison
of metastatic liver tumors before and after treatment with D&V@FOLactis and
RT, or (K) with RT alone at an equivalent dose in patient 2. (M) Hypopharynx
tumors before and after combined treatment in patient 3 using MRI and CT
scans, respectively. (N) Subpleural tumors before and after RT in patient 3.
(O) The levels of cytokines before and after treatment. (The assessments of
the three patients were measured at 1 day before treatment, and at 25 days,
42 days and 7 days post treatment, respectively).cGAS-STING, cyclic GMP-AMP
and synthase-stimulator of Interferon Genes ; ICD, immunogenic cell death;
IFN, interferon; IL, interleukin; DCs, dendritic cells; RT, radiotherapy;
TNF, tumor necrosis factor; Treg, regulatory T cell.
[168]Figure 7
[169]Open in a new tab
Clinical outcomes and immune responses in patients treated with D&V@FOLactis
We then initiated a clinical trial to evaluate the antitumor efficacy
and safety of D&V@FOLactis in patients receiving D&V@FOLactis and RT.
We hypothesized that the combination treatment could synergistically
initiate specific antitumor responses. All enrolled patients
intratumorally injected with two circles of D&V@FOLactis (comprising
four injections) over a 2-month period, with the treatment schedule
adjusted based on monitoring for adverse events. At the time of data
cut-off, three patients completed a two-circle treatment regimen. Two
experienced fever and vomiting following intratumoral injection with
D&V@FOLactis, which were primarily associated with bacterial component.
However, the conditions of these patients improved after the immediate
administration of antipyretics. Subsequently, as a precautionary
measure, antipyretics were applied prior to treatment, and no adverse
effects were observed following the injections. During the follow-up
period, no severe adverse effects were reported in this trial,
indicating that multiple doses of D&V@FOLactis were well-tolerated and
that adverse effects were controllable.
Patient 1, with malignant perineal tumors, was treated with
D&V@FOLactis (2.5×10^9 colony-forming unit (CFU) per injection,
containing 2.5×10^9 CFU FOLactis, 7.5 mg DDP and 0.75 mg VCR) in
combination with RT (plan gross tumor volume, 5 Gy/f×3 f, planning
target volume, planning target volume (PTV), 3 Gy/f×3 f) for perineal
lesions ([170]figure 7F). The combined treatment reduced the length of
perineal tumors from 40.09 mm to 27.08 mm ([171]figure 7G), resulting
in a partial response, whereas there was no significant tumor
suppression in inguinal lymph node lesions treated with an equivalent
dose of RT alone ([172]figure 7H). And the level of squamous cell
carcinoma antigen (SCCA) dramatically decreased from 26.5 ng/mL to
9.41 ng/mL ([173]online supplemental figure S12A). Patient 2, with
stage IV descending colon cancer, received D&V@FOLactis (2.5×10^9 CFU
per injection) along with concurrent RT (first section: 8 Gy×3 f,
second section: 8 Gy×1 f) for liver metastases ([174]figure 7I). The
combined treatment suppressed tumor growth from 6.53 mm to 3.73 mm
within a month post-treatment ([175]figure 7J). In contrast, the volume
of metastatic tumor treated with RT alone exhibited no significant
changes during the observation period (from 14.64 mm to 13.02 mm)
([176]figure 7K). And after two-circle treatment, the tumor biomarkers
remained within the normal range and exhibited no significant abnormal
fluctuations ([177]online supplemental figure S12B). Patient 3, with a
malignant hypopharyngeal tumor, received D&V@FOLactis (2.5×10^9 CFU per
injection) and RT (PTV: 5 Gy×3 f) for hypopharynx tumors ([178]figure
7L). After receiving the combined therapy, the tumor size decreased
from 39.60 mm × 26.12 mm to 34.05 mm × 25.41 mm ([179]figure 7M),
whereas the subpleural lesion treated with RT alone exhibited a slight
increase in size ([180]figure 7N). Additionally, the level of SCCA
experienced a substantial decline from 9.85 ng/mL to 3.04 ng/mL
([181]online supplemental figure S12C). These findings demonstrated
that D&V@FOLactis could inhibit tumors that were refractory to RT,
providing an alternative combined therapeutic approach in clinical
practice (p=0.0719) ([182]online supplemental figure S13). We further
assessed the serum cytokines in peripheral blood to evaluate the
systemic inflammation responses stimulated by D&V@FOLactis. The results
revealed that the levels of cytokines in the serum were all within the
normal range, indicating that intratumoral injection with D&V@FOLactis
would not substantially trigger excessive systemic immune responses or
cytokine storm ([183]figure 7O). In conclusion, the combination of
D&V@FOLactis with RT suggested a possible reduction in multiple types
of tumors with biosafety and presented promising application prospects.
Discussion
ISV uses localized therapeutic approaches to transform tumors into
immunotherapeutic agents, ensuring antigen exposure to initiate
specific immune responses. In previous research, we designed an
engineered FOLactis to deliver Flt3L-OX40L and improve TME. However, it
was not sufficient to evoke tumor cell death and antigen exposure. To
augment tumor-specific immune efficacy, we developed biomineralized
probiotics D&V@FOLactis, combining chemotherapy-induced ICD with
engineered probiotics. This dual-action approach allowed the drugs to
directly kill tumor cells while also acting as immunological
activators, compensating for the insufficient exposure of neoantigens.
As an all-in-one delivery approach, D&V@FOLactis precisely targets
tumors and rapidly achieves optimal outcomes by initiating sequential
chemo-immunotherapy in TME, making them suitable for clinical
application.
D&V@FOLactis effectively activates APCs and elicits T-cell responses
through metalloimmunotherapy. Among existing metal mineralization
technologies, Mg^2+ stands out in all critical aspects. First, Mg^2+
enhances STING activation, which is accompanied by TLR stimulation by
FOLactis.[184]^38 DEG analysis demonstrated that related genes were
upregulated by D&V@FOLactis, marking the effective co-regulation to
elicit pro-inflammatory responses ([185]figure 7E). Next, Mg^2+
promotes T-cell responses through the Mg^2+-LFA-1 axis, strengthening
specific immunity with memory responses.[186]^35 Collectively, Mg^2+ is
crucial for initiating antigen presentation and cytotoxic T-cell
responses. Additionally, clinical safety is another pivotal
consideration. Mg-MOFs were prepared using clinically approved
magnesium sulfate injection in an all-aqueous phase, avoiding high
energy consumption and minimizing reagent residues. In summary, this
biomineralization approach is suitable for clinical translation and
facilitates standardized management.
Improving TME is essential in attacking metastatic tumors that are
inaccessible to ISV. In clinical trial, the combination of D&V@FOLactis
with RT could promptly elicit systemic immune responses. Considering
the adverse effects of high-dose and multiple administrations of RT, we
then investigated the synergistic effect between D&V@FOLactis (5×10^8
CFU) and a single low-dose RT (6 Gy) in bilateral 4T1-model
([187]online supplemental figure S14). The highest inhibition rate
(73.2%) in primary tumors was found in the combined treatment group
([188]online supplemental figure S15A). Simultaneously, with the
further exposure of antigens through radio-based killing effect, the
combined treatment optimized abscopal effect to suppress distant tumors
([189]online supplemental figure S15B) and prolonged survival time
([190]online supplemental figure S16) in the long run. Due to its
potent immunoregulation, the combined applications with D&V@FOLactis
may extend beyond RT to other treatments, including programmed cell
death protein-1 antibodies, immune checkpoint blockade, etc.
In summary, we developed novel biomineralized probiotics D&V@FOLactis
for sequential activation in TME. Nonetheless, D&V@FOLactis was
insufficient to achieve satisfactory systemic therapy outcomes. We
hypothesize that the optimal ratio of D&V and FOLactis may influence
the priming and boosting of immune responses. It is essential to
evaluate the best amounts of drugs and FOLactis to potentiate the
abscopal effect. Furthermore, we explored its combination with RT,
which only modestly increased the abscopal effect, probably due to the
non-selective impact of RT on immune cells. We plan to determine the
optimal sequencing timing for co-administration treatments, ensuring a
superimposing activation effect in TME. And we further initiated a
clinical trial to evaluate the synergistic antitumor effect of
D&V@FOLactis and RT in patients. Although we demonstrated that the
combined treatment was feasible for inhibiting tumors that were
comparatively unresponsive to RT alone, the trial has only enrolled
three patients thus far. Additionally, it lacks long-term follow-up
periods and regular assessment schemes. Therefore, a large randomized
clinical trial is needed in the future to validate the antitumor effect
and safety of D&V@FOLactis. Notably, D&V@FOLactis were demonstrated to
prolong retention time compared with pure FOLactis ([191]figure 3A).
This was probably attributed to the protective Mg-MOFs on its surface,
which shield it from the immune system,[192]^39 and the
gastrointestinal environment.[193]^40 This characteristic was
advantageous for developing oral or intravenous delivery methods.
Further studies are warranted to test our hypothesis regarding the
suitability of D&V@FOLactis for various delivery routes. This may
compensate for the suboptimal abscopal effect and potentially be
applied in some tumors that are surgeon-inaccessible.
Conclusion
We developed biomineralized D&V@FOLactis to integrate ICD with
engineered probiotics. Mg^2+ played an essential role in enhancing the
antigen presentation process and eliciting T-cell responses,
synergizing with FOLactis through STING and TLRs pathways. On
intratumoral administration, D&V@FOLactis reversed the
immunosuppressive TME and inhibited both “cold” and “hot” tumors. In a
clinical trial, the combination of D&V@FOLactis with RT ensured the
further comprehensively reinforced antitumor immunity in patients.
Collectively, we successfully biomineralized probiotics by Mg-MOFs for
the first time and created versatile platforms for the co-delivery of
ISV-based chemo-immunotherapy.
Methods
Bacterial strains, cells, materials and animals
FOLactis was synthesized in our laboratory and statically incubated in
M17 (Difco) medium containing chloramphenicol resistance and 0.5% (w/v)
glucose (GM17) for 18 hours at 30°C. L. lactis NZ9000 and pNZ8148
vector were purchased from MoBiTec (Germany). Murine breast cancer
cells 4T1 and murine colon cancer cells CT26 were obtained from the
Cell Bank of Shanghai Institute of Biochemistry and Cell Biology and
cultured in Roswell Park Memorial Institute (RPMI)-1640, enriched with
10% of fetal calf serum and 1% penicillin and streptomycins in a 5%
CO[2] atmosphere at 37°C. 2-MIM, DDP, and VCR were purchased from
Aladdin (Shanghai, China). Mg sulfate injections were obtained from
Yangzhou Zhongbao Pharmaceutical (Jiangsu, China). BALB/c mice were
ordered from Shanghai Sippr-BK laboratory animal (Shanghai, China). And
all animal procedures were performed in accordance with the guidelines
approved by the Laboratory Animal Care and Use Committee of the
Affiliated Nanjing Drum Tower Hospital of Nanjing University Medical
School.
Synthesis and characterizations of D&V@FOLactis
FOLactis (5×10^8 CFU) was dispersed in 2-MIM solution (3,500 µmol),
stirred at room temperature for 10 min, and centrifuged at 5,000 rpm
for 10 min. Sediment was successively supplemented with VCR, DDP
solution, and MgSO[4] (50 µmol), and stirred for 20 min. D&V@FOLactis
was collected by centrifugating at 5,000 rpm for 10 min. The sizes and
zeta potentials were detected by Zetasizer Nano (Malvern, UK). To
determine the EE, D&V@FOLactis was centrifuged and the concentrations
of DDP and VCR in the supernatant were determined using inductively
coupled plasma mass spectrometry and high-performance liquid
chromatography, respectively.
EE (%) =
[MATH: M−(V×C)
M :MATH]
×100%
(Where M represents the total amounts of drugs added to the reaction
system, V represents the volume of supernatant solution, and C
represents the concentrations of drugs in supernatant solution.)
In vitro drug release of D&V@FOLactis
D&V@FOLactis was dispersed in solutions of 4T1, BMDCs or splenocytes.
At each time point, the suspension was centrifuged, 200 µL release
buffer was collected for evaluation, and fresh buffer was added
immediately.
Cumulative release rate (%) =
[MATH: Cn×V1+∑(Cn-1×
V2)M :MATH]
(Where C[n] represents the concentration of drugs. V[1] and V[2]
represent the volumes of the total release buffer and the replacement
buffer, respectively. M represents the amounts of drugs in
D&V@FOLactis.).
In vitro cytotoxicity assays
Cytotoxicity and biocompatibility were assessed using CCK-8 kits
(Biosharp, China). 4T1 (3,500 cells well^−1) or HUVECs (5,000 cells
well^−1) were seeded in 96-well plates, treated with FOLactis, D&V, and
D&V@FOLactis for 48 hours. After rinsing, the medium was refreshed and
10 µL CCK-8 solution was added per well. The absorbance was measured at
450 nm using a microplate reader.
Cell viability (%) =
[MATH: AX−<
mi
mathvariant="normal">A0<
/msub>A1<
/msub>−A0<
/msub> :MATH]
×100%
(Where A[X] represents the absorbance of cells with drugs, A[1]
represents the absorbance of cells without drugs (ie, in medium only),
and A[0] represents the absorbance of medium.).
Cellular internalization
BMDCs were extracted from the bones of Balb/c mice and cultured in
6-well plates (4×10^6 cells well^−1) in RPMI-1640 medium containing
IL-4 (10 ng/mL^−1, PeproTech, USA) and GM-CSF (20 ng/mL^−1, Xiamen
Amoytop Biotech, China) for 6 days. Differentiated DCs were seeded in 5
cm dishes (1×10^6) for 48 hours and exposed to DIO-stained D&V@FOLactis
(5×10^7) at 37°C. After 2 hours, the adherent DCs were collected,
gently rinsed, stained with Dil and imaged using CLSM.
ICD analysis
4T1 were cultured in 24-well plates (3×10^4 cells well^−1) for
24 hours, then treated with FOLactis, D&V, or D&V@FOLactis for
48 hours. Both suspension and adherent cells were collected, labeled
with anti-CRT primary antibody (1:50) and fluorescein isothiocyanate
(FITC)-conjugated secondary antibody (1:50) successively, and detected
by flow cytometry.
In vitro BMDC stimulation
BMDCs were differentiated for 6 days. On day 6, BMDCs were seeded in
96-well plates (2×10^5 cells well^−1) for 2 days and then co-cultured
with NS, FOLactis (2×10^6 CFU 100 μL^−1), D&V (DDP: 0.12 µg 100 μL^−1;
VCR: 0.012 µg 100 μL^−1), D+V+FOLactis (0.12 µg + 0.012 µg + 2×10^6 CFU
100 μL^−1) and D&V@FOLactis (2×10^6 CFU 100 μL^−1) for 48 hours.
Subsequently, BMDCs were stained with anti-CD11c-FITC, anti-CD80-APC,
and anti-CD86-PE and evaluated by flow cytometry. To simulate TME, 4T1
(1×10^4 cells well^−1) were incubated with BMDCs on day 6, and the
subsequent analysis followed the same procedures.
In vivo biodistribution
4T1-bearing mice were intratumorally injected with DIR-stained FOLactis
or DIR-stained D&V@FOLactis. At predetermined time points, mice were
scanned and the images were captured using CRi Maestro In Vivo Imaging
System (Cambridge Research & Instrumentation, Massachusetts, USA).
Meanwhile, mice were sacrificed, and tumors, TDLNs and major organs
were harvested for ex vivo imaging.
In vivo antitumor therapy and immunization analysis
CT26 (2×10^6) or 4T1 (3×10^5) were subcutaneously implanted into Balb/c
mice (5–6 weeks) at the flank. When the tumor volumes reached 80 mm^3,
mice were randomly divided and intratumorally injected with NS,
FOLactis (5×10^8), D&V (DDP: 30 µg; VCR: 3 µg), or D&V@FOLactis
(5×10^8). Mice were sacrificed when the volumes exceeded 1,500 mm^3.
4T1-bearing mice were sacrificed on day 20. Tumors, TDLNs, and spleens
were collected and digested into single-cell suspension, stained with
anti-CD11c-FITC, anti-CD80-APC, anti-CD86-PE, anti-CD3-FITC,
anti-CD4-BV421, anti-CD8-PC5.5 for flow cytometry analysis. Major
organs were examined by H&E staining, and some tumors were harvested
for immunofluorescence analysis (Wuhan Boerfu Biotechnology, Wuhan,
China).
Tumor volume=L×W×W/2
(Where L and W represent the maximal length and width of the tumor,
respectively).
Tumor suppression rate (%) =
[MATH: VC-VXVC :MATH]
×100%
(Where V[C] and V[X] represent the mean tumor volume in NS group and
treatment group, respectively).
Analysis of chronological changes in TME
4T1 (3×10^5) were subcutaneously injected into Balb/c mice at the
flank. Mice received a single injection of NS, FOLactis (5×10^8), D&V
(DDP: 30 µg; VCR: 3 µg), and D&V@FOLactis (5×10^8 CFU). Subsequently,
tumors and TDLNs were collected at each time point, digested, and
stained for CRT, DCs, and CD8^+ T cells, then analyzed by flow
cytometry.
In vivo abscopal effect
4T1 (3×10^5) were subcutaneously injected into the right flank of
Balb/c mice to create primary tumors. After 3 days, the same cell
density was injected to the left flank to establish distant tumors.
Mice were divided into five groups: NS, FOLactis, D&V, D+V+FOLactis,
and D&V@FOLactis. All treatments were administered to primary tumors.
On day 20, mice from NS and D&V@FOLactis groups were sacrificed. Tumors
and TDLNs were harvested, digested into single-cell suspensions, and
stained with anti-CD3-FITC, anti-CD8-PC5.5, anti-IFN-γ-APC,
anti-CD3-FITC, anti-CD8-PC5.5, anti-CD44-PE, anti-CD62L-APC for flow
cytometry analysis.
Bulk RNA-seq data preprocessing and pathway analysis
Mice were euthanized and submitted for RNA extractions, quality control
analysis, library preparation, and sequencing. Differential expression
analysis was conducted using the “edgeR” R package. The standards
defined as DGEs were absolute value of log2 fold change ≥1 and p
value≤0.05. The protein–protein interaction networks were constructed
in the Search Tool for the Retrieval of Interacting Genes (STRING)
database ([194]https://cn.string-db.org/), with the minimum required
interaction score was 0.900. Then the networks were visualized in
Cytoscape software (V.3.10.2), and the degrees were quantified by the
number of edges among genes. GO and KEGG analyses were applied for DEGs
to explore the potential enriched pathways, using “clusterProfiler” R
package. Pathways with p values≤0.05 were considered significant.
Study design and patients
The clinical trial enrolled patients from Nanjing Drum Tower Hospital
and in accordance with the Declaration of Helsinki and Good Clinical
Practice. Patients were aged between 18 and 80 years with an Eastern
Cooperative Oncology Group performance status of 0–2. Patients had
recurrent or metastatic solid tumors that were either progressing or
intolerant to standard treatment, as determined by pathological
assessments. Furthermore, patients exhibited ≤5 measurable lesions
according to Response Evaluation Criteria In Solid Tumors (RECIST)
V.1.1 criteria and were eligible for intratumoral injection. All
patients provided written informed consent before enrollment.
Patients with a single lesion: intratumoral injection of D&V@FOLactis
was administered.
Patients with two lesions: one lesion received intratumoral injection
of D&V@FOLactis combined with RT, while the other lesion received an
equivalent dose of RT alone.
Patients with ≥2 lesions: one lesion was treated with RT, one lesion
was monitored without intervention, and the remaining lesions were
treated with D&V@FOLactis in combination with RT across multiple
courses, as determined by the investigators.
Statistical analysis
All data were analyzed using GraphPad Prism V.6 and were presented as
mean±SEM. Student’s t-test was used for comparing two groups, and
two-way analyses of variance were performed for comparing tumor
volumes. For survival analysis, log-rank (Mantel-Cox) tests were used.
Supplementary material
online supplemental file 1
[195]jitc-13-8-s001.docx^ (18.7MB, docx)
DOI: 10.1136/jitc-2025-011799
Footnotes
Funding: This work was financially supported by Distinguished Young
Scholars of Jiangsu Province (No. BK20230001), the National Natural
Science Foundation of China (No. 82473182), Jiangsu Provincial Medical
Key Discipline (ZDXK202233), Nanjing Medical Key Laboratory of
Oncology, Jiangsu Province Key Research and Development Program
(BE2023654) and Nanjing Jiangbei New Area key research and development
program.
Provenance and peer review: Not commissioned; externally peer reviewed.
Patient consent for publication: Informed consent was obtained from all
patients in the clinical trial.
Ethics approval: The clinical trial was adhered to the principles
outlined in the Declaration of Helsinki and Good Clinical Practice and
informed consent was obtained from all patients. Ethical committee
approval was obtained at the Ethics Committee of Nanjing Drum Tower
Hospital, Affiliated Hospital of Medical School, Nanjing University and
each participating site (No. 2024-531-01).
Data availability free text: The datasets used and analyzed during the
current study are available from the corresponding author on reasonable
request.
Data availability statement
Data are available upon reasonable request.
References