Little is known about the transcriptomic plasticity and adaptive mechanisms of circulating tumor cells (CTCs) during hematogeneous dissemination. Here we interrogate the transcriptome of 113 single CTCs from 4 different vascular sites, including hepatic vein (HV), peripheral artery (PA), peripheral vein (PV) and portal vein (PoV) using single-cell full-length RNA sequencing in hepatocellular carcinoma (HCC) patients. We reveal that the transcriptional dynamics of CTCs were associated with stress response, cell cycle and immune-evasion signaling during hematogeneous transportation. Besides, we identify chemokine CCL5 as an important mediator for CTC immune evasion. Mechanistically, overexpression of CCL5 in CTCs is transcriptionally regulated by p38-MAX signaling, which recruites regulatory T cells (Tregs) to facilitate immune escape and metastatic seeding of CTCs. Collectively, our results reveal a previously unappreciated spatial heterogeneity and an immune-escape mechanism of CTC, which may aid in designing new anti-metastasis therapeutic strategies in HCC.

Background & Aims: Circulating tumor cells (CTCs) are precursors of cancer metastasis. However, how CTCs evade immunosurveillance during hematogenous dissemination remains unclear. Approach & Results: We identified CTC-platelet adhesions by single-cell RNA sequencing and multiplex immunofluorescence of blood samples from multiple cancer types. Clinically, CTC-platelet aggregates were associated with significantly shorter progression-free survival and overall survival in hepatocellular carcinoma patients. In vitro, ex vivo, and in vivo assays demonstrated direct platelet adhesions gifted cancer cells with an evasive ability from natural killer (NK) cell killing by upregulating inhibitory checkpoint CD155, therefore facilitating distant metastasis. Mechanistically, CD155 was transcriptionally regulated by the FAK/JNK/c-Jun cascade in a platelet contact-dependent manner. Further competition assays and cytotoxicity experiments revealed that CD155 on CTCs inhibited NK cell cytotoxicity only by engaging with immune receptor TIGIT, but not CD96 and DNAM1, another two receptors for CD155. Interrupting the CD155-TIGIT interactions with a TIGIT antibody restored NK cell immunosurveillance on CTCs and markedly attenuated tumor metastasis. Conclusions: Our results demonstrated CTC evasion from NK cell-mediated innate immunosurveillance mainly via immune checkpoint CD155-TIGIT, potentially offering an immunotherapeutic strategy for eradicating CTCs. Export

Background and Aims: Portal vein tumor thrombus (PVTT) worsens the prognosis of hepatocellular carcinoma by increasing intrahepatic dissemination and inducing portal vein hypertension. However, the immune characteristics of PVTT remain unclear. Therefore, this study aims to explore the immune microenvironment in PVTT. Approach and Results: Time-of-flight mass cytometry revealed that macrophages and monocytes were the dominant immune cell type in PVTT, with a higher proportion than in primary tumor and blood (54.1% vs. 26.3% and 9.1%, p<0.05). The differentially enriched clustering of inhibitory and regulatory immune cells in PVTT indicated an immune-suppressive environment. According to the single-cell RNA sequencing, TAM-C5AR1 was characterized by leukocyte chemotaxis and was the most common subpopulation in PVTT (36.7%). Multiplex fluorescent immunohistochemistry staining showed that the C5aR⁺ TAM/Mφ were enriched in PVTT compared to both the primary tumor and liver and positively correlated with C5a (r=0.559, p<0.001). Notably, THP-1 (monocyte cell line) was recruited by CSQT2 (PVTT cell line) and exhibited up-regulation of CD163, CD206, and PD-L1 upon stimulation. C5aR antagonist could reverse this. C5aR⁺ TAMs could also inhibit Granzyme B in CD8⁺ T cells. High infiltration of C5aR⁺ TAMs in PVTT correlated with poor differentiation (p<0.009) and was a risk factor for overall survival (p=0.003) and for reformation of PVTT after resection (p=0.007). Conclusions: TAMs, especially C5aR⁺ TAMs, were enriched in PVTT. C5aR⁺ TAMs contribute to the development of PVTT and poor prognosis by reshaping the immunosuppressive environment.

B cells engage in anti-tumor immunity but how they contribute to cancer suppression remains unclear. We report that inhibiting plasma cell differentiation either in IgMi mice lacking Igh elements needed for antibody secretion or in mice with B cell-specific knockout of Blimp-1 (Blimp-1 BcKO) promotes rather than inhibits antitumor immunity and increases numbers of activated B cells. Deficiency of Blimp-1 in tumor-infiltrating B cells generates a unique transcription profile associated with expansion of mutated clones targeting cognate tumor cells. Major histocompatibility complex class II (MHC II) is required for anti-tumor efficacy. Blimp-1-deficient B cells have increased expression of CD80 and CD86 costimulatory molecules that enhance effector T cell function. The Blimp-1 inhibitor valproic acid suppresses tumor growth in a B cell-dependent manner. Thus, inhibition of plasma cell differentiation results in enhanced tumor-specific antigen presentation by B cells and thereby tumor repression, suggesting a potential avenue of immunotherapy against cancer.