Cell-in-cell (CIC) structures contribute to tumor aggressiveness and poor prognosis in
oral squamous cell carcinoma (OSCC). In vitro 3D models may contribute to the understanding of
the underlying molecular mechanisms of these events. We employed a spheroid model to study
the CIC structures in OSCC. Spheroids were obtained from OSCC (HSC3) and cancer-associated
fibroblast (CAF) lines using the Nanoshuttle-PLTM bioprinting system (Greiner Bio-One). Spheroid
form, size, and reproducibility were evaluated over time (EvosTM XL; ImageJ version 1.8). Slides
were assembled, stained (hematoxylin and eosin), and scanned (Axio Imager Z2/VSLIDE) using
the OlyVIA System (Olympus Life Science) and ImageJ software (NIH) for cellular morphology and
tumor zone formation (hypoxia and/or proliferative zones) analysis. CIC occurrence, complexity, and
morphology were assessed considering the spheroid regions. Well-formed spheroids were observed
within 6 h of incubation, showing the morphological aspects of the tumor microenvironment, such
as hypoxic (core) and proliferative zone (periphery) formation. CIC structures were found in both
homotypic and heterotypic groups, predominantly in the proliferative zone of the mixed HSC3/CAF
spheroids. “Complex cannibalism” events were also noted. These results showcase the potential of
this model in further studies on CIC morphology, formation, and relationship with tumor prognosis
