Two-Photon and Second-Harmonic Imaging of Matrix Remodeling in Breast Cancer Spheroids

Cancer cells accumulate more iron than normal cells to support proliferation. Tumors meet this demand in part by regulating iron transporters such as transferrin receptor (TfR) and divalent metal transporter 1 (DMT1). Our work in triple-negative breast cancer (TNBC) revealed a counterintuitive result: loss of DMT1 increased cancer cell invasion and metastatic outgrowth. We subsequently found that DMT1 loss disrupts iron handling, induces endoplasmic reticulum (ER) stress, and impairs collagen synthesis and extracellular matrix (ECM) organization. These findings prompted us to examine how matrix changes appear within intact tumor spheroids and relate to invasive outgrowth.

In this webinar, Asif shows how he and his fellow researchers developed a two-photon imaging workflow for MDA-MB-231 spheroids grown by liquid overlay with wild-type, DMT1 knockout, or DMT1 overexpressing cells. Two-photon fluorescence imaging reveals spheroid architecture through ZsGreen and DAPI image stacks, maximum intensity projections, orthogonal slices, and surface renderings. Second-harmonic generation (SHG) visualizes fibrillar collagen without a collagen label. The images show that DMT1 knockout spheroids lose their compact structure and have lower SHG intensity than wild-type spheroids. He will also present recent experiments in which treatment with the ER stress relief agent 4-PBA increased SHG intensity and reduced invasive outgrowth.

Alongside these findings, he will discuss spheroid preparation, acquisition decisions, and how the team distinguished SHG signal from fluorescence. Early imaging attempts and the approaches that worked will illustrate how the team established this application on the newly installed microscope at the Albany Medical College Imaging Core.

About the presenter
Arun Asif, Ph.D., is a postdoctoral fellow in the Department of Molecular and Cellular Physiology at Albany Medical College in Albany, N.Y., where he works with Dr. Margarida Barroso on NCI-funded research into iron handling, organelle interactions, and matrix remodeling in triple-negative breast cancer. He received his doctorate from Jeju National University in South Korea, where his research centered on biomedical engineering: 3D cell culture, tumor spheroid models, and organ-on-a-chip systems with integrated biosensors. Before joining Albany Medical College, he held a postdoctoral position at Columbia University, working on the oral microbiome and gastrointestinal cancer organoids.

Asif has published 14 peer-reviewed papers and has an h-index of 11. He is guest-editing a special issue on the organelle interactome for the Journal of Histochemistry and Cytochemistry. He chaired a session at the 2025 American Society for Matrix Biology Biennial Meeting and received the Albany Medical College Postdoctoral Discovery Award in 2026. He has mentored more than 30 trainees. His current work combines confocal, two-photon, and label-free optical imaging with 3D invasion models and cohort-scale transcriptomic and proteomic analysis to understand how metal ion transport shapes the tumor microenvironment.