A multidimensional platform of patient-derived tumors identifies drug susceptibilities for clinical lenvatinib resistance.

Lenvatinib, a second-generation multi-receptor tyrosine kinase inhibitor approved by the FDA for first-line treatment of advanced liver cancer, facing limitations due to drug resistance. Here, we applied a multidimensional, high-throughput screening platform comprising patient-derived resistant liver tumor cells (PDCs), organoids (PDOs), and xenografts (PDXs) to identify drug susceptibilities for conquering lenvatinib resistance in clinically relevant settings. Expansion and passaging of PDCs and PDOs from resistant patient liver tumors retained functional fidelity to lenvatinib treatment, expediting drug repurposing screens. Pharmacological screening identified romidepsin, YM155, apitolisib, NVP-TAE684 and dasatinib as potential antitumor agents in lenvatinib-resistant PDC and PDO models. Notably, romidepsin treatment enhanced antitumor response in syngeneic mouse models by triggering immunogenic tumor cell death and blocking the EGFR signaling pathway. A combination of romidepsin and immunotherapy achieved robust and synergistic antitumor effects against lenvatinib resistance in humanized immunocompetent PDX models. Collectively, our findings suggest that patient-derived liver cancer models effectively recapitulate lenvatinib resistance observed in clinical settings and expedite drug discovery for advanced liver cancer, providing a feasible multidimensional platform for personalized medicine.

A T Cell-Engaging Tumor Organoid Platform for Pancreatic Cancer Immunotherapy.

Pancreatic ductal adenocarcinoma (PDA) is a clinically challenging disease with limited treatment options. Despite a small percentage of cases with defective mismatch DNA repair (dMMR), PDA is included in the most immune-resistant cancer types that are poorly responsive to immune checkpoint blockade (ICB) therapy. To facilitate drug discovery combating this immunosuppressive tumor type, a high-throughput drug screen platform is established with the newly developed T cell-incorporated pancreatic tumor organoid model. Tumor-specific T cells are included in the pancreatic tumor organoids by two-step cell packaging, fully recapitulating immune infiltration in the immunosuppressive tumor microenvironment (TME). The organoids are generated with key components in the original tumor, including epithelial, vascular endothelial, fibroblast and macrophage cells, and then packaged with T cells into their outside layer mimicking a physical barrier and enabling T cell infiltration and cytotoxicity studies. In the PDA organoid-based screen, epigenetic inhibitors ITF2357 and I-BET151 are identified, which in combination with anti-PD-1 based therapy show considerably greater anti-tumor effect. The combinatorial treatment turns the TME from immunosuppressive to immunoactive, up-regulates the MHC-I antigen processing and presentation, and enhances the effector T cell activity. The standardized PDA organoid model has shown great promise to accelerate drug discovery for the immunosuppressive cancer.

Microfluidics guided by deep learning for cancer immunotherapy screening.

Immunocyte infiltration and cytotoxicity play critical roles in both inflammation and immunotherapy. However, current cancer immunotherapy screening methods overlook the capacity of the T cells to penetrate the tumor stroma, thereby significantly limiting the development of effective treatments for solid tumors. Here, we present an automated high-throughput microfluidic platform for simultaneous tracking of the dynamics of T cell infiltration and cytotoxicity within the 3D tumor cultures with a tunable stromal makeup. By recourse to a clinical tumor-infiltrating lymphocyte (TIL) score analyzer, which is based on a clinical data-driven deep learning method, our platform can evaluate the efficacy of each treatment based on the scoring of T cell infiltration patterns. By screening a drug library using this technology, we identified an epigenetic drug (lysine-specific histone demethylase 1 inhibitor, LSD1i) that effectively promoted T cell tumor infiltration and enhanced treatment efficacy in combination with an immune checkpoint inhibitor (anti-PD1) in vivo. We demonstrated an automated system and strategy for screening immunocyte-solid tumor interactions, enabling the discovery of immuno- and combination therapies.

An organoid-based screen for epigenetic inhibitors that stimulate antigen presentation and potentiate T-cell-mediated cytotoxicity.

In breast cancer, genetic heterogeneity, the lack of actionable targets and immune evasion all contribute to the limited clinical response rates to immune checkpoint blockade therapy. Here, we report a high-throughput screen based on the functional interaction of mouse- or patient-derived breast tumour organoids and tumour-specific cytotoxic T cells for the identification of epigenetic inhibitors that promote antigen presentation and potentiate T-cell-mediated cytotoxicity. We show that the epigenetic inhibitors GSK-LSD1, CUDC-101 and BML-210, identified by the screen, display antitumour activities in orthotopic mammary tumours in mice, that they upregulate antigen presentation mediated by the major histocompatibility complex class I on breast tumour cells and that treatment with BML-210 substantially sensitized breast tumours to the inhibitor of the checkpoint programmed death-1. Standardized measurements of tumour-cell killing activity facilitated by tumour-organoid-T-cell screens may help with the identification of candidate immunotherapeutics for a range of cancers.

Atractylenolide I enhances responsiveness to immune checkpoint blockade therapy by activating tumor antigen presentation.

One of the primary mechanisms of tumor cell immune evasion is the loss of antigenicity, which arises due to lack of immunogenic tumor antigens as well as dysregulation of the antigen processing machinery. In a screen for small-molecule compounds from herbal medicine that potentiate T cell-mediated cytotoxicity, we identified atractylenolide I (ATT-I), which substantially promotes tumor antigen presentation of both human and mouse colorectal cancer (CRC) cells and thereby enhances the cytotoxic response of CD8+ T cells. Cellular thermal shift assay (CETSA) with multiplexed quantitative mass spectrometry identified the proteasome 26S subunit non-ATPase 4 (PSMD4), an essential component of the immunoproteasome complex, as a primary target protein of ATT-I. Binding of ATT-I with PSMD4 augments the antigen-processing activity of immunoproteasome, leading to enhanced MHC-I-mediated antigen presentation on cancer cells. In syngeneic mouse CRC models and human patient-derived CRC organoid models, ATT-I treatment promotes the cytotoxicity of CD8+ T cells and thus profoundly enhances the efficacy of immune checkpoint blockade therapy. Collectively, we show here that targeting the function of immunoproteasome with ATT-I promotes tumor antigen presentation and empowers T cell cytotoxicity, thus elevating the tumor response to immunotherapy.

MAL2 drives immune evasion in breast cancer by suppressing tumor antigen presentation.

Immune evasion is a pivotal event in tumor progression. To eliminate human cancer cells, current immune checkpoint therapy is set to boost CD8+ T cell-mediated cytotoxicity. However, this action is eventually dependent on the efficient recognition of tumor-specific antigens via T cell receptors. One primary mechanism by which tumor cells evade immune surveillance is to downregulate their antigen presentation. Little progress has been made toward harnessing potential therapeutic targets for enhancing antigen presentation on the tumor cell. Here, we identified MAL2 as a key player that determines the turnover of the antigen-loaded MHC-I complex and reduces the antigen presentation on tumor cells. MAL2 promotes the endocytosis of tumor antigens via direct interaction with the MHC-I complex and endosome-associated RAB proteins. In preclinical models, depletion of MAL2 in breast tumor cells profoundly enhanced the cytotoxicity of tumor-infiltrating CD8+ T cells and suppressed breast tumor growth, suggesting that MAL2 is a potential therapeutic target for breast cancer immunotherapy.

RNA-binding protein RALY reprogrammes mitochondrial metabolism via mediating miRNA processing in colorectal cancer.

OBJECTIVE: Dysregulated cellular metabolism is a distinct hallmark of human colorectal cancer (CRC). However, metabolic programme rewiring during tumour progression has yet to be fully understood. DESIGN: We analysed altered gene signatures during colorectal tumour progression, and used a complex of molecular and metabolic assays to study the regulation of metabolism in CRC cell lines, human patient-derived xenograft mouse models and tumour organoid models. RESULTS: We identified a novel RNA-binding protein, RALY (also known as hnRNPCL2), that is highly associated with colorectal tumour aggressiveness. RALY acts as a key regulatory component in the Drosha complex, and promotes the post-transcriptional processing of a specific subset of miRNAs (miR-483, miR-676 and miR-877). These miRNAs systematically downregulate the expression of the metabolism-associated genes (ATP5I, ATP5G1, ATP5G3 and CYC1) and thereby reprogramme mitochondrial metabolism in the cancer cell. Analysis of The Cancer Genome Atlas (TCGA) reveals that increased levels of RALY are associated with poor prognosis in the patients with CRC expressing low levels of mitochondrion-associated genes. Mechanistically, induced processing of these miRNAs is facilitated by their N6-methyladenosine switch under reactive oxygen species (ROS) stress. Inhibition of the m6A methylation abolishes the RALY recognition of the terminal loop of the pri-miRNAs. Knockdown of RALY inhibits colorectal tumour growth and progression in vivo and in organoid models. CONCLUSIONS: Collectively, our results reveal a critical metabolism-centric role of RALY in tumour progression, which may lead to cancer therapeutics targeting RALY for treating CRC.

ST2 as checkpoint target for colorectal cancer immunotherapy.

Immune checkpoint blockade immunotherapy delivers promising clinical results in colorectal cancer (CRC). However, only a fraction of cancer patients develop durable responses. The tumor microenvironment (TME) negatively impacts tumor immunity and subsequently clinical outcomes. Therefore, there is a need to identify other checkpoint targets associated with the TME. Early-onset factors secreted by stromal cells as well as tumor cells often help recruit immune cells to the TME, among which are alarmins such as IL-33. The only known receptor for IL-33 is stimulation 2 (ST2). Here we demonstrated that high ST2 expression is associated with poor survival and is correlated with low CD8+ T cell cytotoxicity in CRC patients. ST2 is particularly expressed in tumor-associated macrophages (TAMs). In preclinical models of CRC, we demonstrated that ST2-expressing TAMs (ST2+ TAMs) were recruited into the tumor via CXCR3 expression and exacerbated the immunosuppressive TME; and that combination of ST2 depletion using ST2-KO mice with anti-programmed death 1 treatment resulted in profound growth inhibition of CRC. Finally, using the IL-33trap fusion protein, we suppressed CRC tumor growth and decreased tumor-infiltrating ST2+ TAMs. Together, our findings suggest that ST2 could serve as a potential checkpoint target for CRC immunotherapy.

Heterozygous deletion of chromosome 17p renders prostate cancer vulnerable to inhibition of RNA polymerase II.

Heterozygous deletion of chromosome 17p (17p) is one of the most frequent genomic events in human cancers. Beyond the tumor suppressor TP53, the POLR2A gene encoding the catalytic subunit of RNA polymerase II (RNAP2) is also included in a ~20-megabase deletion region of 17p in 63% of metastatic castration-resistant prostate cancer (CRPC). Using a focused CRISPR-Cas9 screen, we discovered that heterozygous loss of 17p confers a selective dependence of CRPC cells on the ubiquitin E3 ligase Ring-Box 1 (RBX1). RBX1 activates POLR2A by the K63-linked ubiquitination and thus elevates the RNAP2-mediated mRNA synthesis. Combined inhibition of RNAP2 and RBX1 profoundly suppress the growth of CRPC in a synergistic manner, which potentiates the therapeutic effectivity of the RNAP2 inhibitor, α-amanitin-based antibody drug conjugate (ADC). Given the limited therapeutic options for CRPC, our findings identify RBX1 as a potentially therapeutic target for treating human CRPC harboring heterozygous deletion of 17p.

The expression and significance of microRNA in different stages of colorectal cancer.

BACKGROUND: The aim of this study is to compare microRNA expression patterns in different stages of colorectal cancer (CRC) and to discuss the significance of the application of microRNAs in the clinical treatment of CRC. METHODS: The study used gene chip technology to analyze genetic sequences in CRC tissues and surrounding normal tissues at different cancer stages. The bioinformatics profiles of the target genes of the different microRNAs were analyzed to clarify the target gene-related pathways and their functions in the disease. RESULTS: A total of 368 target genes with differential expression, including 275 upregulated and 93 downregulated genes, were screened from CRC patients in different stages of the disease. These microRNAs participated widely in the occurrence and development processes of CRC. The microRNA expression profiles obviously differed in tissues at different CRC stages. CONCLUSION: microRNA regulation of CRC samples can be used as a tool to control the occurrence and development of tumor cells.

Rapid mixing of viscous liquids by electrical coiling.

The control for the processing of precursor liquids determines whether the properties and functions of the final material product can be engineered. An inherent challenge of processing viscous liquids arises from their large resistance to deform. Here, we report on the discovery of an electric approach that can significantly contribute to address this challenge. The applied electric force can induce a straight viscous jet to coil, and the resulting coiling characteristics are governed by the electric stress. We demonstrate the promising use of electrical coiling in the rapid and efficient mixing of viscous liquids. Remarkably, the degree of mixing can be precisely adjusted by tuning the applied electric stress. Our approach of controlling the coiling electrically has important implications on applications such as dispensing and printing of resins, printing patterned surfaces and scaffolds, processing of food and generating non-woven fabrics.

AFM nanoindentation detection of the elastic modulus of tongue squamous carcinoma cells with different metastatic potentials.

UNLABELLED: Although significant advances have been made in understanding the molecular mechanisms that influence tongue squamous cell carcinoma (TSCC) metastasis, less is known about the association between the cellular elastic modulus and TSCC metastasis. Atomic force microscopy (AFM) nanoindentation via the rate-jump method was used to detect the elastic modulus of TSCC cells from patients and cell lines with different metastatic potentials. TSCC cells with higher metastatic potential showed decreases in the elastic modulus compared to TSCC cells with lower metastatic potential. Moreover, the decrease in elastic modulus was accompanied with epithelial-mesenchymal transition (EMT), cytoskeleton (F-actin and ß-tubulin) changes, small nucleus size and large nucleus/cytoplasm (N/C) ratio. The present findings demonstrate a close relationship between the cellular elastic modulus and the metastasis of TSCC. The elastic modulus detected by AFM nanoindentation via the rate-jump method can potentially be used to grade the metastatic potential of TSCC. FROM THE CLINICAL EDITOR: This team of investigators report the use of an atomic force microscopy-based method to determine the elastic modulus of tongue squamous cell carcinoma cells, and demonstrate that such cells with higher metastatic potential show decreased elastic modulus compared to cells with lower metastatic potential.

[Transient expression in microplasmodia of Physarum polycephalum].

The plasmodium of Physarum polycephalum is a suitable eukaryotic cell for cell cycle investigation, but there is no compatible transient expression system for the plasmodium. Using the promoter and terminator of ardC actin of Physarum polycephalum substituted the CMV IE and SV40 polyA of plasmid pDsRedl-N1, using cassette PardC-MCS-DsRed1-TardC substituted the cassette PardC-hph-TardC of plasmid pTB38, we constructed plasmids pXM1 and pXM2 for transient expression of red fluorescent protein (RFP) in Physarum polycephalum respectively. After reconstituting the transcription elongation factor homologous gene (pelf1) of Physarum polycephalum into the pXM2, we generated a plasmid pXM2-pelf1. After the plasmid pXM1, pXM2 and pXM2-pelf1 were electroporated into the plasmodium of Physarum polycephalum, we observed optimum RFP and PELF1-RFP expression under fluoroscope and confocal microscope between 24-48 h after electroporation, and found that ELF1-RFP expression was accumulated in nucleus of microplasmodium, the optimum electroporation parameters were 40 V/cm electric field, 1 ampere current, and 70 micros electric shock time. The results suggest that this expression system is qualified for transient expression of specific protein in plasmodium of Physarum polycephalum.

A novel Physarum polycephalum SR protein kinase specifically phosphorylates the RS domain of the human SR protein, ASF/SF2.

A 1591-bp cDNA of a serine-rich protein kinase (SRPK)-like protein has been identified in Physarum polycephalum (GenBank accession No. DQ140379). The cDNA contains two repeat sequences at bp 1-153 and bp 395-547. The encoding sequence is 56% homologous to human SRPK1 and is named Physarum SRPK (PSRPK). Consistent with other SRPKs, the consensus motifs of PSRPK are within the two conserved domains (CDs). However, divergent motifs between the N-terminal and CDs are much shorter than the corresponding sequences of other SRPKs. To study the structure and function of this protein, we performed co-expression experiment in Escherichia coli and in vitro phosphorylation assay to investigate the phosphorylation effect of recombinant PSRPK on the human SR protein, ASF/SF2. Western blot analysis showed that PSRPK could phosphorylate ASF/SF2 in E. coli cells. Autoradiographic examination showed that both recombinant PSRPK and a truncated form of PSRPK with a 28-aa deletion at the N-terminus could phosphorylate ASF/SF2 and a truncated form of ASF/SF2 that contains the RS domain. However, these two forms of PSRPK could not phosphorylate a truncated form ASF/SF2 that lacks the RS domain. A truncated form of PSRPK that lacks either of CDs does not have any phosphorylation activity. These results indicated that, like other SRPKs, the phosphorylation site in PSRPK is located within the RS domain of the SR protein and that its phosphorylation activity is closely associated with the two CDs. This study on the structure and function of PSRPK demonstrates that it is a new member of the SRPK family.

Identification of a nuclear localization motif in the serine/arginine protein kinase PSRPK of physarum polycephalum.

BACKGROUND: Serine/arginine (SR) protein-specific kinases (SRPKs) are conserved in a wide range of organisms, from humans to yeast. Studies showed that SRPKs can regulate the nuclear import of SR proteins in cytoplasm, and regulate the sub-localization of SR proteins in the nucleus. But no nuclear localization signal (NLS) of SRPKs was found. We isolated an SRPK-like protein PSRPK (GenBank accession No. DQ140379) from Physarum polycephalum previously, and identified a NLS of PSRPK in this study. RESULTS: We carried out a thorough molecular dissection of the different domains of the PSRPK protein involved in its nuclear localization. By truncation of PSRPK protein, deletion of and single amino acid substitution in a putative NLS and transfection of mammalian cells, we observed the distribution of PSRPK fluorescent fusion protein in mammalian cells using confocal microscopy and found that the protein was mainly accumulated in the nucleus; this indicated that the motif contained a nuclear localization signal (NLS). Further investigation with truncated PSPRK peptides showed that the NLS (318PKKGDKYDKTD328) was localized in the alkaline Omega-loop of a helix-loop-helix motif (HLHM) of the C-terminal conserved domain. If the 318PKKGDK322 sequence was deleted from the loop or K320 was mutated to T320, the PSRPK fluorescent fusion protein could not enter and accumulate in the nucleus. CONCLUSION: This study demonstrated that the 318PKKGDKYDKTD328 peptides localized in the C-terminal conserved domain of PSRPK with the Omega-loop structure could play a crucial role in the NLS function of PSRPK.

[Development of time-resolved fluoroimmunoassay kit for detection of IgM antibodies against hepatitis B core antigen].

OBJECTIVE: To prepare a time-resolved fluoroimmunoassay (TRFIA) kit for clinical detection of IgM antibodies to hepatitis B core antigen (HBc). METHODS: Immunocapture method was used to develop the TRFIA kit for detection of the anti-HBc IgM antibodies, and the precision, cross-reactivity and sensitivity of the kit were tested with the clinical serum samples. RESULTS: The intra- and inter-assay coefficients of variation of the TRFIA kit were 4.8%-7.2% and 7.5%-8.6%, respectively, and no cross-reactivity with anti-HBs, anti-HBc-IgG or anti-HBe was found. Comparison of the results of the TRFIA kit and enzyme-linked immunosorbent assay (ELISA) demonstrated greater sensitivity of the kit than ELISA in detecting the anti-HBc IgM antibodies in 584 serum samples. According to the detection results in 300 serum samples from healthy donors, the cutoff value of the TRFIA kit was 4.5 times of the fluorescence value of the negative control. CONCLUSION: This TRFIA kit for detecting anti-HBc IgM antibodies meets the demand for clinical application and can replace the ELISA kits.