Assistant Professor University of Texas at Dallas Richardson, Texas, United States
Introduction: : Colorectal cancer (CRC) remains one of the most common causes of cancer-related deaths in the United States and worldwide [1]. Over the past decades, public awareness has led to increased rates of routine colonoscopy screening, thereby causing a decline in the incidence of average onset colorectal cancer (AO CRC), which affects patients above 50 years of age. Meanwhile, early onset colorectal cancer (EO CRC), which affects patients under 50 years of age, has been rising at an alarming 2% per year and is expected to increase by 124% by the year 2030 [2]. The precise etiology of EO CRC remains unknown, with no unique mutational drivers. Epidemiological studies have indicated that chronic, low-grade inflammation could represent a pathogenic mechanism in the EO CRC patient cohort [3]. It is well established that collagen remodeling and overall tissue stiffening are hallmarks of inflammation and subsequent fibrosis. However, there is a lack of biomechanical and mechanobiological data on EO CRC. Therefore, we hypothesized that stiffening of colonic tissues may represent a quantifiable hallmark of EO CRC.
Materials and
Methods: : We tested this hypothesis using colonic tissue specimens harvested from patients undergoing CRC surgical resection. We analyzed 19 AO CRC patients and 14 EO CRC patients and for each patient we obtained tissue from the primary tumor and from matched normal tissue (retrieved at least 5 cm away from the tumor). For each sample we punched two cylindrical specimens (3 mm in diameter) and subjected them to four local indentation tests and to one global unconfined compression test to determine their material properties [4]. We used established methods [6] to quantify collagen microstructure via histology and quantified collagen content (via Masson’s Trichrome staining), collagen organization (via Picrosirius Red staining), and collagen architecture (via multiphoton Second Harmonic Generation imaging).
Results, Conclusions, and Discussions:: Local (mesoscale) indentation revealed that EO tissues are materially stiffer than their AO counterparts. Global (macroscale) compression data analyzed using a nonlinear viscoelastic model of tissue behavior [5] confirmed the increase in tissue stiffness and further showed that EO tissues are more viscous. Overall, biomechanical data suggest that EO CRC tissues undergo fibrotic remodeling. Immunofluorescence (IF) staining showed that these patterns of matrix remodeling were associated with an increase in Vimentin expression, consistent with a larger stromal cell compartment in EO tissues. Since YAP is a mechanosensitive effector of the Hippo signaling pathway, which regulates organ size and cell growth [8], we examined epithelial cell proliferation via immunohistochemistry (IHC) and found that EO tissues had elevated nuclear Ki67 with respect to AO tissues. For the purpose of in vitro validation, 2D polyacrylamide (PA) gels designed to match the stiffness of human CRC tissues were functionalized with 1.5% Matrigel and used to culture the colon cancer cell lines HT29 and SW480. After 72 hours in culture, epithelial morphology and proliferation were quantified by measuring, respectively, the cell aspect ratio from Phalloidin staining and the fraction of EdU positive cells. Finally, epithelial organoids were established from a subset of patient samples with AO and EO CRC as described previously [9]. Such patient-derived organoids were encapsulated in 3D Tyramine-Substituted Hyaluronate (HA) gels of varying stiffness and cultured overnight. We used a commercial assay (CellTiter-Glo® 3D) to assess differences in 3D organoid proliferation. In vitro culture of colon cancer cell lines on 2D PA gels and colonic organoids in 3D HA gels revealed a stiffness-dependent increase in cell proliferation. Therefore, by combining multiscale biomechanical testing, quantitative histology, IF/IHC, and in vitro modeling, we find that fibrotic remodeling in the colonic submucosa is associated with increased epithelial proliferation in the colonic mucosa, thereby supporting the notion that a stiffer tissue microenvironment may play an important role in EO CRC initiation.
Acknowledgements and/or References (Optional): : [1] Siegel R.L. et al., CA Cancer J Clin, 73: 233-254, 2023. [2] Hofseth L.J.H. et al., Nat Rev Gastroenterol Hep, 17: 352-364, 2020. [3] Murphy C.C. et al., Gastroenterology, 156: 958-965, 2019. [4] Kang W., Ferruzzi J. et al., iScience, 24: 103252, 2021. [5] Tuttle T. et al., J Mech Behav Biomed Mater, 143: 105926, 2023. [6] Ferruzzi J. et al., Biomech Model Mechanobiol, 17: 1281-1295, 2018. [7] Merritt C.R. et al., Nat Biotechnol, 38: 586-599, 2020. [8] Piccolo S. et al., Physiol Rev, 94: 1287-1312, 2014. [9] Sato T. et al., Gastroenterology, 141: 1762-1772, 2011.
