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We have successfully established murine and human 3dimensional co-culture models of primary liver tumorderived organoids with cancer-associated fibroblasts. This model system enables the study of the interactions between tumor cells and the stromal compartment and the response to anticancer drugs.BACKGROUND & AIMS: Cancer-associated fibroblasts (CAFs) play a key role in the cancer process, but the research progress is hampered by the paucity of preclinical models that are essential for mechanistic dissection of cancer cell-CAF interactions. Here, we aimed to establish 3-dimensional (3D) organotypic co-cultures of primary liver tumor-derived organoids with CAFs, and to understand their interactions and the response to treatment. METHODS:Liver tumor organoids and CAFs were cultured from murine and human primary liver tumors. 3D co-culture models of tumor organoids with CAFs and Transwell culture systems were established in vitro. A xenograft model was used to investigate the cell-cell interactions in vivo. Gene expression analysis of CAF markers in our hepatocellular carcinoma cohort and an online liver cancer database indicated the clinical relevance of CAFs. RESULTS:To functionally investigate the interactions of liver cancer cells with CAFs, we successfully established murine and human 3D co-culture models of liver tumor organoids with CAFs. CAFs promoted tumor organoid growth in co-culture with direct cell-cell contact and in a Transwell system via paracrine signaling. Vice versa, cancer cells secrete paracrine factors regulating CAF physiology. Co-transplantation of CAFs with liver tumor organoids of mouse or human origin promoted tumor growth in xenograft models. Moreover, tumor organoids conferred resistance to clinically used anticancer drugs including sorafenib, regorafenib, and 5-fluorouracil in the presence of CAFs, or the conditioned medium of CAFs. CONCLUSIONS:We successfully established murine and human 3D co-culture models and have shown robust effects of CAFs in liver cancer nurturing and treatment resistance.

Purpose: Liver metastasis develops in >50% of patients with colorectal cancer (CRC), and is a leading cause of CRC-related mortality. We aimed to identify which inhibitory immune checkpoint pathways can be targeted to enhance functionality of intra-tumoral T-cells in mismatch repair-proficient liver metastases of colorectal cancer (LM-CRC). Methodology: Intra-tumoral expression of multiple inhibitory molecules was compared among mismatch repair-proficient LM-CRC, peritoneal metastases of colorectal cancer (PM-CRC) and primary CRC. Expression of inhibitory molecules was also analyzed on leukocytes isolated from paired resected metastatic liver tumors, tumor-free liver tissues, and blood of patients with mismatch repair-proficient LM-CRC. The effects of blocking inhibitory pathways on tumor-infiltrating T-cell responses were studied in ex vivo functional assays. Results: Mismatch repair-proficient LM-CRC showed higher expression of inhibitory receptors on intra-tumoral T-cells and contained higher proportions of CD8+ T-cells, dendritic cells and monocytes than mismatch repair-proficient primary CRC and/or PM-CRC. Inhibitory receptors LAG3, PD-1, TIM3 and CTLA4 were higher expressed on CD8+ T-cells, CD4+ T-helper and/or regulatory T-cells in LM-CRC tumors compared with tumor-free liver and blood. Antibody blockade of LAG3 or PD-L1 increased proliferation and effector cytokine production of intra-tumoral T-cells isolated from LM-CRC in response to both polyclonal and autologous tumor-specific stimulations. Higher LAG3 expression on intra-tumoral CD8+ T-cells associated with longer progression-free survival of LM-CRC patients. Conclusion: Mismatch repair-proficient LM-CRC may be more sensitive to immune checkpoint inhibitors than mismatch repair-proficient primary CRC. Blocking LAG3 enhances tumor-infiltrating T-cell responses of mismatch repair-proficient LM-CRC, and therefore may be a new promising immunotherapeutic target for LM-CRC.

Cancer stem cells (CSCs) or tumor-initiating cells (TICs) are thought to be the main drivers for disease progression and treatment resistance across various cancer types. Identifying and targeting these rare cancer cells, however, remains challenging with respect to therapeutic benefit. Here, we report the enrichment of LGR5 expressing cells, a well-recognized stem cell marker, in mouse liver tumors, and the upregulation of LGR5 expression in human hepatocellular carcinoma. Isolated LGR5 expressing cells from mouse liver tumors are superior in initiating organoids and forming tumors upon engraftment, featuring candidate TICs. These cells are resistant to conventional treatment including sorafenib and 5-FU. Importantly, LGR5 lineage ablation significantly inhibits organoid initiation and tumor growth. The combination of LGR5 ablation with 5-FU, but not sorafenib, further augments the therapeutic efficacy in vivo. Thus, we have identified the LGR5 + compartment as an important TIC population, representing a viable therapeutic target for combating liver cancer.

CD8 þ TILs that contain terminally exhausted PD1 high CD8 þ cells generally respond to ex vivo single PD1 blockade, whereas CD8 þ TILs of most HCC patients without this subset do not respond to single PD1 blockade but can be functionally restored by ex vivo co-blockade of TIGIT and PD1. BACKGROUND & AIMS:TIGIT is a co-inhibitory receptor, and its suitability as a target for cancer immunotherapy in HCC is unknown. PD1 blockade is clinically effective in about 20% of advanced HCC patients. Here we aim to determine whether coblockade of TIGIT/PD1 has added value to restore functionality of HCC tumor-infiltrating T cells (TILs).METHODS: Mononuclear leukocytes were isolated from tumors, paired tumor-free liver tissues (TFL) and peripheral blood of HCC patients, and used for flow cytometric phenotyping and functional assays. CD3/CD28 T-cell stimulation and antigen-specific assays were used to study the ex vivo effects of TIGIT/PD1 single or dual blockade on T-cell functions.RESULTS: TIGIT was enriched, whereas its co-stimulatory counterpart CD226 was down-regulated on PD1 high CD8 þ TILs. PD1 high TIGIT þ CD8 þ TILs co-expressed exhaustion markers TIM3 and LAG3 and demonstrated higher TOX expression. Furthermore, this subset showed decreased capacity to produce IFN-g and TNF-a. Expression of TIGIT-ligand CD155 was up-regulated on tumor cells compared with hepatocytes in TFL. Whereas single PD1 blockade preferentially enhanced ex vivo functions of CD8 þ TILs from tumors with PD1 high CD8 þ TILs (high PD1 expressers), coblockade of TIGIT and PD1 improved proliferation and cytokine production of CD8 þ TILs from tumors enriched for PD1 int CD8 þ TILs (low PD1 expressers). Importantly, ex vivo co-blockade of TIGIT/PD1 improved proliferation, cytokine production, and cytotoxicity of CD8 þ TILs compared with single PD1 blockade.CONCLUSIONS: Ex vivo, co-blockade of TIGIT/PD1 improves functionality of CD8 þ TILs that do not respond to single PD1 blockade. Therefore co-blockade of TIGIT/PD1 could be a promising immune therapeutic strategy for HCC patients.

Rationale: Regulatory T cells (Treg) play a pivotal role in the immunosuppressive tumor micro-environment in cancer, including mesothelioma. Recently, the combination of autologous tumor lysate-pulsed dendritic cells (DC) and metronomic cyclophosphamide (mCTX) was reported as a feasible and well-tolerated treatment in malignant pleural mesothelioma patients and further as a method to reduce circulating Tregs.Objectives: The aim of this study was to establish the immunological effects of mCTX alone and in combination with DC-based immunotherapy on circulating Treg and other T cell subsets in mesothelioma patients.Methods: Ten patients received mCTX and DC-based immunotherapy after chemotherapy (n = 5) or chemotherapy and debulking surgery (n = 5). Peripheral blood mononuclear cells before, during and after treatment were analyzed for various Treg and other lymphocyte subsets by flow cytometry.Results: After one week treatment with mCTX, both activated FoxP3hi and naïve CD45RA+ Tregs were effectively decreased in all patients. In addition, a shift from naïve and central memory towards effector memory and effector T cells was observed. Survival analysis showed that overall Treg levels before treatment were not correlated with survival, however, nTreg levels before treatment were positively correlated with survival. After completion of mCTX and DC-based immunotherapy treatment, all cell subsets returned to baseline levels, except for the proportions of proliferating EM CD8 T cells, which increased.Conclusions: mCTX treatment effectively reduced the proportions of circulating Tregs, both aTregs and nTregs, thereby favoring EM T cell subsets in mesothelioma patients. Interestingly, baseline levels of nTregs were positively correlated to overall survival upon complete treatment.

No curative treatment options are available for advanced hepatocellular carcinoma (HCC). Anti‐PD1 antibody therapy can induce tumor regression in 20% of advanced HCC patients, demonstrating that co‐inhibitory immune checkpoint blockade has therapeutic potential for this type of cancer. However, whether agonistic targeting of co‐stimulatory receptors might be able to stimulate anti‐tumor immunity in HCC is as yet unknown. We investigated whether agonistic targeting of the co‐stimulatory receptor GITR could reinvigorate ex vivo functional responses of tumor‐infiltrating lymphocytes (TIL) freshly isolated from resected tumors of HCC patients. In addition, we compared GITR expression between TIL and paired samples of leukocytes isolated from blood and tumor‐free liver tissues, and studied the effects of combined GITR and PD1 targeting on ex vivo TIL responses. In all three tissue compartments, CD4 + FoxP3 + regulatory T cells (Treg) showed higher GITR − expression than effector T‐cell subsets. The highest expression of GITR was found on CD4 + FoxP3 hi CD45RA − activated Treg in tumors. Recombinant GITR‐ligand as well as a humanized agonistic anti‐GITR antibody enhanced ex vivo proliferative responses of CD4 + and CD8 + TIL to tumor antigens presented by mRNA‐transfected autologous B‐cell blasts, and also reinforced proliferation, IFN‐γ secretion and granzyme B production in stimulations of TIL with CD3/CD28 antibodies. Combining GITR ligation with anti‐PD1 antibody nivolumab further enhanced tumor antigen‐specific responses of TIL in some, but not all, HCC patients, compared to either single treatment. In conclusion, agonistic targeting of GITR can enhance functionality of HCC TIL, and may therefore be a promising strategy for single or combinatorial immunotherapy in HCC.

The use of cytokines for immunotherapy shows clinical efficacy but is frequently accompanied by severe adverse events caused by excessive and systemic immune activation. Here, we set out to address these challenges by engineering a fusion protein of a single, potency-reduced, IL15 mutein and a PD1-specific antibody (anti-PD1-IL15m). This immunocytokine was designed to deliver PD1-mediated, avidity-driven IL2/15 receptor stimulation to PD1+ tumor-infiltrating lymphocytes (TIL) while minimally affecting circulating peripheral natural killer (NK) cells and T cells. Treatment of tumor-bearing mice with a mouse cross-reactive fusion, anti-mPD1–IL15m, demonstrated potent antitumor efficacy without exacerbating body weight loss in B16 and MC38 syngeneic tumor models. Moreover, anti-mPD1–IL15m was more efficacious than an IL15 superagonist, an anti-mPD-1, or the combination thereof in the B16 melanoma model. Mechanistically, anti-PD1–IL15m preferentially targeted CD8+ TILs and single-cell RNA-sequencing analyses revealed that anti-mPD1–IL15m treatment induced the expansion of an exhausted CD8+ TIL cluster with high proliferative capacity and effector-like signatures. Antitumor efficacy of anti-mPD1–IL15m was dependent on CD8+ T cells, as depletion of CD8+ cells resulted in the loss of antitumor activity, whereas depletion of NK cells had little impact on efficacy. The impact of anti-hPD1–IL15m on primary human TILs from patients with cancer was also evaluated. Anti-hPD1–IL15m robustly enhanced the proliferation, activation, and cytotoxicity of CD8+ and CD4+ TILs from human primary cancers in vitro, whereas tumor-derived regulatory T cells were largely unaffected. Taken together, our findings showed that anti-PD1–IL15m exhibits a high translational promise with improved efficacy and safety of IL15 for cancer immunotherapy via targeting PD1+ TILs. See related Spotlight by Felices and Miller, p. 1110.