A 3D biofabricated cutaneous squamous cell carcinoma tissue model with multi-channel confocal microscopy imaging biomarkers to quantify antitumor effects of chemotherapeutics in tissue

Abstract

Cutaneous squamous cell carcinoma (cSCC) causes approximately 10,000 deaths annually in the U. S. Current therapies are largely ineffective against metastatic and locally advanced cSCC. There is a need to identify novel, effective, and less toxic small molecule cSCC therapeutics. We developed a 3-dimensional bioprinted skin (3DBPS) model of cSCC tumors together with a microscopy assay to test chemotherapeutic effects in tissue. The full thickness SCC tissue model was validated using hematoxylin and eosin (H&E) and immunohistochemical histological staining, confocal microscopy, and cDNA microarray analysis. A nondestructive, 3D fluorescence confocal imaging assay with tdTomato-labeled A431 SCC and ZsGreen-labeled keratinocytes was developed to test efficacy and general toxicity of chemotherapeutics. Fluorescence-derived imaging biomarkers indicated that 50% of cancer cells were killed in the tissue after 1μM 5-Fluorouracil 48-hour treatment, compared to a baseline of 12% for untreated controls. The imaging biomarkers also showed that normal keratinocytes were less affected by treatment (11% killed) than the untreated tissue, which had no significant killing effect. Data showed that 5-Fluorouracil selectively killed cSCC cells more than keratinocytes. Our 3DBPS assay platform provides cellular-level measurement of cell viability and can be adapted to achieve nondestructive high-throughput screening (HTS) in bio-fabricated tissues.

Keywords: 3D printing; confocal microscopy; in vitro model; screening; squamous cell carcinoma.

Figure 1. 3D, Biofabricated tissue model system and chemotherapeutic assay using multi-channel confocal microscopy imaging biomarkers.

From aliquots of cryopreserved cells, full thickness skin samples were biofabricated that mimic normal human skin morphology in vivo. After 4 days of media submersion, cancer spheroids were introduced that displaced and invaded the normal skin constructs. Constructs were transferred to our custom transwell apparatus to form construct arrays. Array cassettes were compatible with both incubation and imaging so they were imaged, treated by addition of 1μM 5FU to their nutrient media and re-imaged after incubation.

Figure 2

Stratum corneum (A), stratum spinosum (B), and dermis (C) of human skin and biofabricated skin at different depth layers in en face, confocal reflectance and fluorescence microscopy. The dermal-epidermal junction is shown as a dotted white line in the standard histology of skin and biofabricated skin images.

Figure 3

(A) H&E, Ki67, and Anti-RFP stained histology of 3DBPS model. (B) 3D confocal image of skin tissue with SCC tumor (purple: phalloidin staining, red: tdTomato red fluorescent protein (RFP) labeled SCC, blue: Hoescht staining). (C) Bimodal en face confocal microscopy of tdT-RFP-labeled cSCC (red arrows) in 3DBPS constructs with and without Zs-GFP-labeled neonatal keratinocytes (green arrow). All cSCC A431 cells are labeled with RFP while only 1% of keratinocytes are labeled with Zs-GFP resulting in sparsity of the green signal. (D) SCC signature genes that are up- and down-regulated in A431 cells grown in vitro and untreated full thickness 3DBPS without SCC spheroids compared to 3DBPS with SCC spheroids. Log2 fold change is normalized to Keratin 10.

Figure 4

(A) Bimodal imaging examples of control and treated tumors (red) before and after the treatment period. cSCC tumors are red, reflectance is grayscale, and keratinocytes are green. All cSCC A431 cells are labeled with RFP while only 1% of keratinocytes are labeled with Zs-GFP resulting in sparsity of the green signal. (B) H&E, Ki67, and Anti-RFP antibody staining of untreated (control) tumor model and tumor model treated with 5FU for 48 hours. (C) 5FU treatment effect on tdT-RFP-labeled cSCC spheroids and Zs-GFP-labeled keratinocytes including p-value and effect size, δ. Two imaging biomarkers are shown, number of segmented objects and total fluorescence of the size-thresholded segmented objects. NOTE: the fluorescence in panel A is intentionally saturated for display.