Substrate Stiffness Modulates the Maturation of Human Pluripotent Stem-Cell-Derived Hepatocytes

Obtaining functional hepatocytes from human pluripotent stem cells (hPSCs) holds great potential for applications in drug safety testing, as well in the field of regenerative medicine. However, developing functionally mature hPSC-derived hepatocytes (hPSC-Heps) remains a challenge. We hypothesized that the cellular microenvironment plays a vital role in the maturation of immature hepatocytes. In this study, we examined the role of mechanical stiffness, a key component of the cellular microenvironment, in the maturation of hPSC-Heps. We cultured hPSC-Heps on collagen-coated polyacrylamide hydrogels with varying elastic moduli. On softer substrates the hPSC-Heps formed compact colonies while on stiffer substrates they formed a diffuse monolayer. We observed an inverse correlation between albumin production and substrate stiffness. The expression of key cytochrome enzymes, which are expressed at higher levels in the adult liver compared to the fetal liver, also correlated inversely with substrate stiffness, whereas fetal markers such as Cyp3A7 and AFP showed no correlation with stiffness. Culture of hPSC-Heps on soft substrates for 12 days led to 10–30 fold increases in the expression of drug-metabolizing enzymes. These results demonstrate that substrate stiffness similar to that of the liver enables aspects of the maturation of hPSC-Heps

Association of <i>ABCB1</i> and <i>FLT3</i> Polymorphisms with Toxicities and Survival in Asian Patients Receiving Sunitinib for Renal Cell Carcinoma

<div><p>Sunitinib is a tyrosine kinase inhibitor used as first-line treatment for metastatic renal cell carcinoma (mRCC). Asian ethnicity has been previously associated with lower clearance and greater toxicities for sunitinib treatment, relative to Caucasian ethnicity. Research focusing on identifying corresponding biomarkers of efficacy and toxicity has been hitherto conducted in Caucasian populations, and few of the reported associations have been externally validated. Our work thus aims to investigate candidate biomarkers in Asian patients receiving sunitinib, comparing the observed genotype effects with those reported in Caucasian populations. Using data from 97 Asian mRCC patients treated with sunitinib, we correlated 7 polymorphisms in <i>FLT3</i>, <i>ABCB1</i>, <i>VEGFR2</i>, <i>ABCG2</i> and <i>BIM</i> with patient toxicities, response, and survival. We observed a stronger association of <i>FLT3 738T</i> genotype with leucopenia in our Asian dataset than that previously reported in Caucasian mRCC patients (odds ratio [OR]=8.0; <i>P</i>=0.03). We observed significant associations of <i>FLT3 738T</i> (OR=2.7), <i>ABCB1 1236T</i> (OR=0.3), <i>ABCB1 3435T</i> (OR=0.1), <i>ABCB1 2677T</i> (OR=0.4), <i>ABCG2 421A</i> (OR=0.3) alleles and <i>ABCB1 3435</i>, <i>1236</i>, <i>2677 TTT</i> haplotype (OR=0.1) on neutropenia. Primary resistance (OR=0.1, <i>P</i>=0.004) and inferior survival (progression-free: hazard ratio [HR]=5.5, <i>P</i>=0.001; overall: HR=5.0, <i>P</i>=0.005) were associated with the <i>ABCB1 3435</i>, <i>1236</i>, <i>2677 TTT</i> haplotype. In conclusion, <i>ABCB1</i> and <i>FLT3</i> polymorphisms may be helpful in predicting sunitinib toxicities, response and survival benefit in Asian mRCC patients. We have also validated the association between <i>FLT3 738T</i> and sunitinib-induced leucopenia previously reported in Caucasian populations, but have not validated other reported genetic associations.</p></div

Survival curves.

<p>(A) Patients grouped according to the <i>ABCB1 3435C/T</i>, <i>1236C/T</i>, <i>2677G/T</i> haplotype; median PFS was 2.4 months for homozygous carriers of the <i>TTT</i> haplotype and 8.4 months for other cases (<i>P</i> = 0.001). (B) Patients grouped according to the <i>ABCB1 3435C/T</i>, <i>1236C/T</i>, <i>2677G/TA</i> haplotype; median OS was 4.6 months for homozygous carriers of the <i>TTT</i> haplotype and 19.6 months for other cases (<i>P</i> = 0.005).</p

Polymorphisms genotyped and allele frequencies.

<p>^a Patients successfully genotyped.</p><p>^b Includes 34 <i>GT</i> and 10 <i>AG</i> individuals.</p><p>^c Includes 2 <i>AA</i>, 12 <i>AT</i> and 13 <i>TT</i> individuals.</p><p>^d A 2,903-bp deletion polymorphism in intron 2 of <i>BIM</i> previously associated with resistance to tyrosine kinase inhibitors [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0134102#pone.0134102.ref028" target="_blank">28</a>]. As we were unable to genotype formalin-fixed tissues with the current method, only 45 patients were typed.</p><p>^e Variant allele frequencies.</p><p>Polymorphisms genotyped and allele frequencies.</p

Patient demographics and baseline characteristics (n = 97).

<p>Patient demographics and baseline characteristics (n = 97).</p

DataSheet1_A comprehensive next generation sequencing tissue assay for Asian-prevalent cancers—Analytical validation and performance evaluation with clinical samples.xlsx

Introduction: A well-validated diagnostic assay with curated biomarkers complements clinicopathological factors to facilitate early diagnosis and ensure timely treatment delivery. This study focuses on an Asian-centric cancer diagnostic assay designed and thoroughly validated against commercially available standard references and a cohort of over 200 clinical specimens spanning 12 diverse Asian-centric cancer types.Methods: The assay uses hybrid-capture probes capable of profiling DNA aberrations from 572 cancer-related genes and 91 RNA fusion partners. The panel can detect clinically-tractable biomarkers such as microsatellite instability (MSI) and tumor mutation burden (TMB).Results: Analytical evaluation demonstrated 100% specificity and 99.9% sensitivity within a ≥5% VAF limit of detection (LoD) for SNV/Indels. RNA-based fusion features an LoD of ≥5 copies per nanogram input when evaluated against commercial references. Excellent linearity and concordance were observed when benchmarking against orthogonal methods in identifying MSI status, TMB scores and RNA fusions. Actionable genetic alterations were identified in 65% of the clinical samples.Conclusion: These results demonstrate a molecular diagnostic assay that accurately detects genomic alterations and complex biomarkers. The data also supports an excellent performance of this assay for making critical diagnoses and well-informed therapeutic decisions in Asian prevalent cancers.</p