Pancreatic ductal adenocarcinoma (PDAC) poses challenges for immunotherapy due to its low immunogenicity and highly immunosuppressive environment. To overcome these issues, Selvanesan, Chandra, and Quispe-Tintaya et al. developed a microbial-based treatment to deliver an immunogenic protein into tumor cells to trigger antitumor responses. Their results were recently published in Science Translational Medicine.

The researchers used the attenuated bacterium Listeria monocytogenes (Listeria) to deliver the immunogenic antigen tetanus toxoid (TT) to tumor cells. They hypothesized that this antigen could reactivate pre-existing memory T cells generated by childhood vaccination. In addition, treatment was supplemented with low-dose gemcitabine (GEM) to limit myeloid-derived suppressor cells (MDSCs) and tumor-associated macrophages (TAMs) in the tumor microenvironment (TME). For their experiments, they used a syngeneic Panc-02 and a transgenic KrasG12D,p53R172H,Pdx1-Cre (KPC) pancreatic tumor model.

Listeria-TT could infect Panc-02 cells in vitro, resulting in TT protein expression in the tumor cells. Quantification of the number of Listeria-TT in various tissues of the Panc-02 model after intraperitoneal (i.p.) injection revealed high concentrations in the primary tumor on day 1, and in pancreatic metastases on day 3. On day 7, bacteria were still detected in metastases, but not in normal tissues. Intravenous delivery led to poor accumulation in tumors or metastases.

To test the infection rate and cell death in human tumor lines, the Mia-PaCa2 cell line was used. In vitro, this line had a 12- to 17-fold higher infection rate than the mouse tumor cells, with a similar killing rate. Furthermore, when Mia-PaCa2 tumors were established in the pancreas of nude mice, a single high dose of Listeria i.p. resulted in Listeria accumulation in the tumor.

To study whether Listeria-TT could be taken up in vivo by tumor cells, Listeria-TT was fluorescently labeled and i.p. injected in transgenic mice with fluorescently labeled orthotopic pancreatic tumors and macrophages. Listeria-TT was found inside tumor cells and macrophages. TT was found in approximately 80% of the tumor and was only detected in mice treated with Listeria-TT +/- GEM, with a higher amount of TT and more Listeria bacteria found in mice treated with Listeria-TT + GEM.

Given that most patients with cancer are older, with a natural reduction in naive T cells, immunotherapy may be more effective when it does not require naive T cell responses. The researchers generated TT-specific memory CD4⁺ and CD8⁺ T cells in mice by injection of TT vaccine two weeks before tumors were inoculated. When tumors were established, KPC mice were treated with Listeria-TT +/- GEM. After treatment, spleens were isolated and restimulated with TT protein in vitro to analyze memory T cell responses. Both CD4⁺ and CD8⁺ T cells were strongly activated and had a high specificity for TT protein. Comparing T cells activated by Listeria carrying the endogenous tumor-associated antigen Survivin with the TT-specific T cells revealed that TT was more immunogenic than Survivin, resulting in more vigorous T cell responses.

Assessing the TME by immunohistochemistry, the researchers found that there was an increase in tumoral CD4⁺ T cells in vaccinated KPC mice that received treatment, particularly in areas expressing TT and CD31⁺ vessels. This increase was higher when Listeria-TT was combined with GEM. CD8⁺ T cells, on the other hand, were only sparsely present in the tumor. Additionally, granzyme B and perforin were detected in these tumors, along with markers for T cell trafficking, CD40, and CD40L.

KPC tumors had large lymph node-like structures (LNS) in their proximity in mice treated with Listeria-TT +/- GEM. These LNS had high perforin and granzyme B production, and most of the T cells found in these areas that produced IFNγ had a memory phenotype (CD44⁺CD62L^-).

RNAseq of tumors from KPC mice treated with Listeria-TT confirmed upregulation of CD4, limited upregulation of CD8, upregulation of multiple granzymes, and – less abundantly – upregulation of perforin. Furthermore, genes associated with apoptotic pathways and MHC-II genes were upregulated after Listeria-TT +/- GEM treatment.

To assess the effects of GEM addition to the Listeria treatment, Panc-02 tumor-bearing mice were treated with GEM. In these mice, MDSCs were reduced by 80-90% in the blood and 50% in tumors, and TAMs reduced by 67%. The remaining MDSCs and TAMs produced fewer immunosuppressive cytokines, while TNFɑ and CD80 expression increased after treatment with Listeria-TT + GEM.

Assessing the efficacy of the treatment, KPC mice were vaccinated with standard TT vaccine and subsequently treated with Listeria-TT + GEM. This resulted in a reduction in the growth and metabolic activity of the tumors. Additionally, in the Panc-02 peritoneal model with early and advanced pancreatic cancer, there was a near-complete elimination of the early-stage tumors, and a significant reduction in tumor burden (primary and metastases) in advanced-stage cancer. In survival studies, the treatment also improved outcomes.

To assess the role of CD4⁺ T cells in the observed antitumor effects, the researchers conducted depletion experiments and showed a significant impact of CD4 depletion and a limited effect of CD8 depletion on tumor growth.

The researchers then aimed to show that TT-specific T cells could destroy TT-expressing tumor cells by stably transfecting TT in Panc-02 cells for in vitro assays. TT-specific memory CD4⁺ T cells were obtained by immunizing C57BL/6 mice with the TT vaccine, and these cells could destroy the TT-expressing Panc-02 cells in vitro.

Comparing Listeria with Listeria-TT treatment revealed that TT was a significant contributor to the treatment, as Listeria-TT treatment reduced tumor burden and metastases significantly compared to Listeria. Similarly, when vaccinated and unvaccinated Panc-02-bearing mice were treated with Listeria-TT, the effect of vaccination was noticeable, resulting in significantly lower tumor weight.

Finally, the researchers assessed the safety of the treatment in various mouse models, and found that the treatment dose was well below the median lethal dose, and that effects on leukocytes and liver function were minimal, indicating safety.

Therefore, this novel immunotherapeutic method that makes use of bacterial tumor-specific delivery of an antigen, to which most people have developed memory T cell immunity through childhood vaccinations, offers great potential for development in poorly immunogenic cancer types.

Write-up by Maartje Wouters, image by Lauren Hitchings