BackgroundTumors drive blood vessel growth to obtain oxygen and nutrients to support tumor expansion, and they also can induce lymphatic vessel growth to facilitate fluid drainage and metastasis. These processes have generally been studied separately, so that it is not known how peritumoral blood and lymphatic vessels grow relative to each other.MethodsThe murine B16-F10 melanoma and chemically-induced squamous cell carcinoma models were employed to analyze large red-colored vessels growing between flank tumors and draining lymph nodes. Immunostaining and microscopy in combination with dye injection studies were used to characterize these vessels.ResultsEach peritumoral red-colored vessel was found to consist of a triad of collecting lymphatic vessel, vein, and artery, that were all enlarged. Peritumoral veins and arteries were both functional, as detected by intravenous dye injection. The enlarged lymphatic vessels were functional in most mice by subcutaneous dye injection assay, however tumor growth sometimes blocked lymph drainage to regional lymph nodes. Large red-colored vessels also grew between benign papillomas or invasive squamous cell carcinomas and regional lymph nodes in chemical carcinogen-treated mice. Immunostaining of the red-colored vessels again identified the clustered growth of enlarged collecting lymphatics, veins, and arteries in the vicinity of these spontaneously arising tumors.ConclusionsImplanted and spontaneously arising tumors induce coordinate growth of blood and lymphatic vessel triads. Many of these vessel triads are enlarged over several cm distance between the tumor and regional lymph nodes. Lymphatic drainage was sometimes blocked in mice before lymph node metastasis was detected, suggesting that an unknown mechanism alters lymph drainage patterns before tumors reach draining lymph nodes.
The MET oncoprotein is a tyrosine kinase that is the only known receptor for its ligand, HGF (Hepatocyte Growth Factor). In approximately 5% of gastric cancers, MET is activated by focal amplification, rendering it constitutively active and ligand independent. MET-amplified cancer cell lines and xenografts are typically dependent on MET signaling for proliferation or survival. To test this dependency in patients, a number of small molecule MET inhibitors, including AMG337, are currently undergoing clinical evaluation for the treatment of MET-amplified tumors.Here we characterize resistance mechanisms to MET inhibitor AMG337 in vitro. We show that under standard culture conditions, resistance does not readily arise in MET-amplified gastric cell lines SNU620 and MKN45 treated with a therapeutic dose (>IC90) of AMG337. Surprisingly however, exogenous recombinant human HGF enables resistance to readily appear, with emergent cultures possessing resistance mutations in the MET activation loop at positions D1228 and Y1230. Phosphoprotein analysis shows that HGF increases autophosphorylation and activation of mutant MET sufficiently to drive cell proliferation even in the presence of AMG337.Notably, deep sequencing and clonal analysis of individually barcoded cells show that pre-existing mutant clones are likely present in cultures prior to drug treatment. However, such clones are unable to robustly proliferate in drug-containing media unless HGF is present. Based on copy number analysis, we propose that a single or small number of pre-existing mutant MET alleles in an individual cell is sufficient to drive resistance in the presence of exogenous HGF. In contrast, in HGF-negative media, a single mutant MET allele is insufficient and thus resistance does not readily arise unless gradual drug dose escalation protocols that select for increased mutant MET copy number are employed.Our results identify a key mechanism by which HGF can promote resistance to MET inhibitors in MET-amplified cancer cell lines. Because HGF is present in tumor microenvironments, combining anti-HGF antibodies with maximally-tolerated doses of small molecule MET inhibitors such as AMG337 may be beneficial in preventing resistance and in promoting durable responses. Citation Format: Marcia Gordon, Alexandra Croft, Chi-Ming Li, Sean Taylor, Sean R. Caenepeel, Kim Quon. HGF promotes resistance to MET inhibitor AMG337 in MET-amplified gastric cancer cells. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr LB-241. doi:10.1158/1538-7445.AM2015-LB-241
Background: Adoptive T-cell therapy with engineered T-cell receptors (eTCR) has demonstrated promising, yet modest clinical benefit to date. Key obstacles for this technology are 1) competition with the endogenous TCR for CD3 components that are required for surface expression of a functional TCR complex and 2) mispairing of endogenous and exogenous TCRα and TCRβ chains. Substituting murine TCR constant domains has been shown to enhance eTCR expression, but increases the risk of immunologic rejection. Knockout or knockdown of the endogenous TCR has also been shown to improve eTCR expression. Current eTCR delivery approaches use semi-randomly integrating lentivirus or retrovirus to generate an eTCR expressing T-cell product. An alternative approach is to combine TCR knockout with targeted integration of eTCRs into the T-cell receptor alpha constant (TRAC) locus. In this study, we evaluate newly discovered eTCRs specific for human papillomavirus (HPV) type 16 oncogenic protein E7, expressed via these various gene engineering approaches, to optimize engineered T-cell functionality. Methods: HPV E7-specific eTCRs were introduced into primary human T-cells by lentiviral transduction or a dual CRISPR-Cas9/AAV eTCR delivery platform for targeted insertion into the TRAC locus using homology directed repair (HDR). Comparisons were made among TCR sufficient and TCR knockouT-cells with gene delivery by lentivirus or HDR. Engineered T-cell function was assessed both in vitro and in vivo against HPV-16+ head and neck squamous cell carcinoma cell lines. Results: CRISPR/Cas9-mediated TRAC editing eliminated endogenous TCR expression in >85% of T-cells. The impact of TRAC editing on eTCR expression and engineered T-cell function was variable across multiple eTCRαβ sequences. With many eTCRs, TRAC editing in lentivirus-derived populations resulted in increased expression and improved cytokine and killing responses. Targeted insertion of these eTCRs into the TRAC locus by HDR increased engineered cell product homogeneity and enhanced function compared to lentiviral integration. However, there were also examples of eTCRs that were minimally impacted by TRAC locus engineering. Conclusions: Our results demonstrate that elimination of the endogenous TCR, alone or combined with targeted insertion at the TRAC locus, improves eTCR expression and engineered T-cell function for the majority of eTCRs tested. While infrequent, some eTCRs were less impacted by TRAC locus engineering, suggesting a sequence-specific property that enables these eTCRs to out-compete the endogenous TCR for CD3 componentry. The cause for this variable response to TRAC locus engineering is still being explored. Citation Format: Alexandra Croft, Cameron Brandt, Stephen Burleigh, Eric Chadwick, Melissa Chin, Dean Toy, Bailey Donahue, Clay Patton, Stephen Goldfless, Brian Belmont, Ruth Salmon, Grant Welstead, Blythe D. Sather, David J. Huss. Targeted insertion of an HPV-16 E7-specific engineered T-cell receptor into the TRAC locus [abstract]. In: Proceedings of the Fourth CRI-CIMT-EATI-AACR International Cancer Immunotherapy Conference: Translating Science into Survival; Sept 30-Oct 3, 2018; New York, NY. Philadelphia (PA): AACR; Cancer Immunol Res 2019;7(2 Suppl):Abstract nr A027.
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