The biological process of lysine-tRNA charging is therapeutically targetable in liver cancer.

BACKGROUND & AIMS: Mature transfer RNAs (tRNA) charged with amino acids decode mRNA to synthesize proteins. Dysregulation of translational machineries has a fundamental impact on cancer biology. This study aims to map the tRNAome landscape in liver cancer patients and to explore potential therapeutic targets at the interface of charging amino acid with tRNA. METHODS: Resected tumour and paired tumour-free (TFL) tissues from hepatocellular carcinoma (HCC) patients (n = 69), and healthy liver tissues from organ transplant donors (n = 21), HCC cell lines, and cholangiocarcinoma (CC) patient-derived tumour organoids were used. RESULTS: The expression levels of different mature tRNAs were highly correlated and closely clustered within individual tissues, suggesting that different members of the tRNAome function cooperatively in protein translation. Interestingly, high expression of tRNA-Lys-CUU in HCC tumours was associated with more tumour recurrence (HR 1.1; P = .022) and worse patient survival (HR 1.1; P = .0037). The expression of Lysyl-tRNA Synthetase (KARS), the enzyme catalysing the charge of lysine to tRNA-Lys-CUU, was significantly upregulated in HCC tumour tissues compared to tumour-free liver tissues. In HCC cell lines, lysine deprivation, KARS knockdown or treatment with the KARS inhibitor cladosporin effectively inhibited overall cell growth, single cell-based colony formation and cell migration. This was mechanistically mediated by cell cycling arrest and induction of apoptosis. Finally, these inhibitory effects were confirmed in 3D cultured patient-derived CC organoids. CONCLUSIONS: The biological process of charging tRNA-Lys-CUU with lysine sustains liver cancer cell growth and migration, and is clinically relevant in HCC patients. This process can be therapeutically targeted and represents an unexplored territory for developing novel treatment strategies against liver cancer.

GITR ligation enhances functionality of tumor-infiltrating T cells in hepatocellular carcinoma.

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+ FoxP3hi 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.

Reduction of immunosuppressive tumor microenvironment in cholangiocarcinoma by ex vivo targeting immune checkpoint molecules.

BACKGROUND & AIMS: Cholangiocarcinoma is an aggressive hepatobiliary malignancy originating from biliary tract epithelium. Whether cholangiocarcinoma is responsive to immune checkpoint antibody therapy is unknown, and knowledge of its tumor immune microenvironment is limited. We aimed to characterize tumor-infiltrating lymphocytes (TILs) in cholangiocarcinoma and assess functional effects of targeting checkpoint molecules on TILs. METHODS: We isolated TILs from resected tumors of patients with cholangiocarcinoma and investigated their compositions compared with their counterparts in tumor-free liver (TFL) tissues and blood, by flow cytometry and immunohistochemistry. We measured expression of immune co-stimulatory and co-inhibitory molecules on TILs, and determined whether targeting these molecules improved ex vivo functions of TILs. RESULTS: Proportions of cytotoxic T cells and natural killer cells were decreased, whereas regulatory T cells were increased in tumors compared with TFL. While regulatory T cells accumulated in tumors, the majority of cytotoxic and helper T cells were sequestered at tumor margins, and natural killer cells were excluded from the tumors. The co-stimulatory receptor GITR and co-inhibitory receptors PD1 and CTLA4 were over-expressed on tumor-infiltrating T cells compared with T cells in TFL and blood. Antagonistic targeting of PD1 or CTLA4 or agonistic targeting of GITR enhanced effector molecule production and T cell proliferation in ex vivo stimulation of TILs derived from cholangiocarcinoma. The inter-individual variations in TIL responses to checkpoint treatments were correlated with differences in TIL immune phenotype. CONCLUSIONS: Decreased numbers of cytotoxic immune cells and increased numbers of suppressor T cells that over-express co-inhibitory receptors suggest that the tumor microenvironment in cholangiocarcinoma is immunosuppressive. Targeting GITR, PD1 or CTLA4 enhances effector functions of tumor-infiltrating T cells, indicating that these molecules are potential immunotherapeutic targets for patients with cholangiocarcinoma. LAY SUMMARY: The defense functions of immune cells are suppressed in cholangiocarcinoma tumors. Stimulating or blocking "immune checkpoint" molecules expressed on tumor-infiltrating T cells can enhance the defense functions of these cells. Therefore, these molecules may be promising targets for therapeutic stimulation of immune cells to eradicate the tumors and prevent cancer recurrence in patients with cholangiocarcinoma.

Systemic T-cell and humoral responses against cancer testis antigens in hepatocellular carcinoma patients.

Hepatocellular carcinoma (HCC) is the fourth leading cause of cancer-related deaths worldwide due to high recurrence rates after curative treatment and being frequently diagnosed at an advanced stage. Immune-checkpoint inhibition (ICPI) has yielded impressive clinical successes in a variety of solid cancers, however results in treatment of HCC have been modest. Vaccination could be a promising treatment to synergize with ICPI and enhance response rates. Cancer testis antigens (CTAs) were recently discovered to be widely expressed in HCC and expression in macroscopically tumor-free tissues correlated with recurrence, implying the presence of micro-satellites. To determine whether CTAs are immunogenic in HCC patients, we analyzed systemic T-cell and humoral responses against seven CTAs in 38 HCC patients using a multitude of techniques; flowcytometry, ELISA and whole antigen and peptide stimulation assays. CTA-specific T-cells were detected in all (25/25) analyzed patients, of which most had a memory phenotype but did not exhibit unequivocal signs of chronic stimulation or recent antigen encounter. Proliferative CD4+ and CD8+ T-cell responses against these CTAs were found in 14/16 analyzed HCC patients. CTA-peptide stimulation-induced granzyme B, IL2, and TNFa in 8/8 analyzed patients, including two MAGEA1 peptides included based on in silico prediction. Finally, IgG responses were observed in 13/32 patients, albeit with low titers. The presence of CD4+ and CD8+ T-cells and IgG responses shows the immunogenicity of these CTAs in HCC-patients. We hypothesize that vaccines based on these tumor-specific antigens may boost preexisting CTA-specific immunity and could enhance therapeutic efficacy of ICPI in advanced HCC.

Detection of oncogenic mutations in paired circulating tumor DNA and circulating tumor cells in patients with hepatocellular carcinoma.

BACKGROUND AND AIMS: Circulating tumor cells (CTCs) or circulating tumor DNA (ctDNA) may be used for diagnostic or prognostic purposes in patients with hepatocellular carcinoma (HCC). We aim to determine whether CTCs or ctDNA are suitable to determine oncogenic mutations in HCC patients. METHODS: Twenty-six mostly advanced HCC patients were enrolled. 30 mL peripheral blood from each patient was obtained. CellSearch system was used for CTC detection. A sequencing panel covering 14 cancer-relevant genes was used to identify oncogenic mutations. TERT promoter C228T and C250T mutations were determined by droplet digital PCR. RESULTS: CTCs were detected in 27% (7/26) of subjects but at low numbers (median: 2 cells, range: 1-15 cells) and ctDNA in 77% (20/26) of patients. Mutations in ctDNA were identified in several genes: TERT promoter C228T (77%, 20/26), TP53 (23%, 6/26), CTNNB1 (12%, 3/26), PIK3CA (12%, 3/26) and NRAS (4%, 1/26). The TERT C228T mutation was present in all patients with one or more ctDNA mutations, or detectable CTCs. The TERT C228T and TP53 mutations detected in ctDNA were present at higher levels in matched primary HCC tumor tissue. The maximal variant allele frequency (VAF) of ctDNA was linearly correlated with largest tumor size and AFP level (Log10). CtDNA (or TERT C228T) positivity was associated with macrovascular invasion, and positivity of ctDNA (or TERT C228T) or CTCs (≥ 2) correlated with poor patient survival. CONCLUSIONS: Oncogenic mutations could be detected in ctDNA from advanced HCC patients. CtDNA analysis may serve as a promising liquid biopsy to identify druggable mutations.

Current Tolerance-Associated Peripheral Blood Gene Expression Profiles After Liver Transplantation Are Influenced by Immunosuppressive Drugs and Prior Cytomegalovirus Infection.

Spontaneous operational tolerance to the allograft develops in a proportion of liver transplant (LTx) recipients weaned off immunosuppressive drugs (IS). Several previous studies have investigated whether peripheral blood gene expression profiles could identify operational tolerance in LTx recipients. However, the reported gene expression profiles differed greatly amongst studies, which could be caused by inadequate matching of clinical parameters of study groups. Therefore, the purpose of this study was to validate differentially expressed immune system related genes described in previous studies that identified tolerant LTx recipients after IS weaning. Blood was collected of tolerant LTx recipients (TOL), a control group of LTx recipients with regular IS regimen (CTRL), a group of LTx recipients with minimal IS regimen (MIN) and healthy controls (HC), and groups were matched on age, sex, primary disease, time after LTx, and cytomegalovirus serostatus after LTx. Quantitative Polymerase Chain Reaction was used to determine expression of twenty selected genes and transcript variants in PBMCs. Several genes were differentially expressed between TOL and CTRL groups, but none of the selected genes were differentially expressed between HC and TOL. Principal component analysis revealed an IS drug dosage effect on the expression profile of these genes. These data suggest that use of IS profoundly affects gene expression in peripheral blood, and that these genes are not associated with operational tolerance. In addition, expression levels of SLAMF7 and NKG7 were affected by prior cytomegalovirus infection in LTx recipients. In conclusion, we found confounding effects of IS regimen and prior cytomegalovirus infection, on peripheral blood expression of several selected genes that were described as tolerance-associated genes by previous studies.

TIGIT and PD1 Co-blockade Restores ex vivo Functions of Human Tumor-Infiltrating CD8+ T Cells in Hepatocellular Carcinoma.

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 co-blockade 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 PD1high CD8+ TILs. PD1high TIGIT+ CD8+ TILs co-expressed exhaustion markers TIM3 and LAG3 and demonstrated higher TOX expression. Furthermore, this subset showed decreased capacity to produce IFN-γ and TNF-α. 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+ TILs from tumors with PD1high CD8+ TILs (high PD1 expressers), co-blockade of TIGIT and PD1 improved proliferation and cytokine production of CD8+ TILs from tumors enriched for PD1int CD8+ TILs (low PD1 expressers). Importantly, ex vivo co-blockade of TIGIT/PD1 improved proliferation, cytokine production, and cytotoxicity of CD8+ TILs compared with single PD1 blockade. CONCLUSIONS: Ex vivo, co-blockade of TIGIT/PD1 improves functionality of CD8+ 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.

Expression of Cancer Testis Antigens in Tumor-Adjacent Normal Liver Is Associated with Post-Resection Recurrence of Hepatocellular Carcinoma.

High recurrence rates after resection of hepatocellular carcinoma (HCC) with curative intent impair clinical outcomes of HCC. Cancer/testis antigens (CTAs) are suitable targets for cancer immunotherapy if selectively expressed in tumor cells. The aims were to identify CTAs that are frequently and selectively expressed in HCC-tumors, and to investigate whether CTAs could serve as biomarkers for occult metastasis. Tumor and paired tumor-free liver (TFL) tissues of HCC-patients and healthy tissues were assessed for mRNA expression of 49 CTAs by RT-qPCR and protein expression of five CTAs by immunohistochemistry. Twelve CTA-mRNAs were expressed in ≥10% of HCC-tumors and not in healthy tissues except testis. In tumors, mRNA and protein of ≥ 1 CTA was expressed in 78% and 71% of HCC-patients, respectively. In TFL, CTA mRNA and protein was found in 45% and 30% of HCC-patients, respectively. Interestingly, CTA-expression in TFL was an independent negative prognostic factor for post-resection HCC-recurrence and survival. We established a panel of 12 testis-restricted CTAs expressed in tumors of most HCC-patients. The increased risk of HCC-recurrence in patients with CTA expression in TFL, suggests that CTA-expressing (pre-)malignant cells may be a source of HCC-recurrence, reflecting the relevance of targeting these to prevent HCC-recurrence.

Cancer-Associated Fibroblasts Provide a Stromal Niche for Liver Cancer Organoids That Confers Trophic Effects and Therapy Resistance.

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.

An Engineered IL15 Cytokine Mutein Fused to an Anti-PD1 Improves Intratumoral T-cell Function and Antitumor Immunity.

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.

FcγRIIB engagement drives agonistic activity of Fc-engineered αOX40 antibody to stimulate human tumor-infiltrating T cells.

BACKGROUND: OX40 (CD134) is a costimulatory molecule of the tumor necrosis factor receptor superfamily that is currently being investigated as a target for cancer immunotherapy. However, despite promising results in murine tumor models, the clinical efficacy of agonistic αOX40 antibodies in the treatment of patients with cancer has fallen short of the high expectation in earlier-stage trials. METHODS: Using lymphocytes from resected tumor, tumor-free (TF) tissue and peripheral blood mononuclear cells (PBMC) of 96 patients with hepatocellular and colorectal cancers, we determined OX40 expression and the in vitro T-cell agonistic activity of OX40-targeting compounds. RNA-Seq was used to evaluate OX40-mediated transcriptional changes in CD4+ and CD8+ human tumor-infiltrating lymphocytes (TILs). RESULTS: Here, we show that OX40 was overexpressed on tumor-infiltrating CD4+ T cells compared with blood and TF tissue-derived T cells. In contrast to a clinical candidate αOX40 antibody, treatment with an Fc-engineered αOX40 antibody (αOX40_v12) with selectively enhanced FcγRIIB affinity, stimulated in vitro CD4+ and CD8+ TIL expansion, as well as cytokine and chemokine secretions. The activity of αOX40_v12 was dependent on FcγRIIB engagement and intrinsic CD3/CD28 signals. The transcriptional landscape of CD4+ and CD8+ TILs shifted toward a prosurvival, inflammatory and chemotactic profile on treatment with αOX40_v12. CONCLUSIONS: OX40 is overexpressed on CD4+ TILs and thus represents a promising target for immunotherapy. Targeting OX40 with currently used agonistic antibodies may be inefficient due to lack of OX40 multimerization. Thus, Fc engineering is a powerful tool in enhancing the agonistic activity of αOX40 antibody and may shape the future design of antibody-mediated αOX40 immunotherapy.

LGR5 marks targetable tumor-initiating cells in mouse liver cancer.

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.

Low-dose cyclophosphamide depletes circulating naïve and activated regulatory T cells in malignant pleural mesothelioma patients synergistically treated with dendritic cell-based immunotherapy.

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.

Blockade of LAG3 enhances responses of tumor-infiltrating T cells in mismatch repair-proficient liver metastases of colorectal cancer.

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.