Characterization of tumor progression in engineered tissue using infrared spectroscopic imaging

Abstract

Engineered tissues can provide models for imaging and disease progression and the use of such models is becoming increasingly prevalent. While structural characterization of these systems is documented, a combination of biochemical and structural knowledge is often helpful. Here, we apply Fourier transform infrared (FT-IR) spectroscopic imaging to examine an engineered tissue model of melanoma. We first characterize the biochemical properties and spectral changes in different layers of growing skin. Second, we introduce malignant melanocytes to simulate tumor formation and growth. Both cellular changes associated with tumor formation and growth can be observed. In particular, chemical changes associated with tumor-stromal interactions are observed during the course of tumor growth and appear to influence a 50-100 microm region. The development of this analytical approach combining engineered tissue with spectroscopy, imaging and computation will allow for quality control and standardization in tissue engineering and novel scientific insight in cancer progression.

Fig. 1

Skin structure and characterizations. (A) Schematic of human (left) and engineered skin (right). The epidermis is located at the skin-air interface while the dermis is located to the bottom of the image and contains various histologic structures. Engineered skin retains the most important components but does not contain accessory structures. (B) Histology of skin is usually deduced manually in tissue stained by Haematoxylin and Eosin (H&E). Epidermis typically stains darker than dermis. (C) Infrared absorbance image from a corresponding serial section of the tissue at 1660 cm⁻¹. Human skin was imaged on a low-e slide in reflectance mode, and engineered skin was imaged on a BaF₂ substrate in transmission mode. (D) IR spectra of single pixels from epidermis, dermis and stratum corneum regions of human and engineered skin after piecewise linear baseline subtraction.

Fig. 2

Melanoma progression in 28 days. Tumors (arrows) grow larger and invade into the dermis over time, as shown by both H&E-stains followed by histological recognition and IR spectroscopic imaging. IR absorbance images at 1660 cm⁻¹ and 1284 cm⁻¹ highlight epidermal and dermal regions, respectively, indicating different chemical compositions between these two regions and their facile delineation by simple spectral metrics.

Fig. 3

Temporal analysis of tumor and normal stroma. (A) Spectra of 10 pixels of tumors (green arrow) and 10 × 10 pixels of normal stroma that are at least 500 μm away from tumors (blue square) are extracted for temporal analysis. (B) Comparison of average spectra from tumor and normal stroma regions. (C) CDM visualization of spectra of normal stroma. The average spectrum is shown above the data and the central data block displays the difference of each pixel spectrum from the average spectrum.

Fig. 4

Temporal behavior of normal and tumor-associated stroma. (A) Spectral regions of tumor (red circle) and tumor associated stroma are extracted for analysis of temporal changes. (B) Spectral analysis of normal and tumor-associated stroma demonstrates likely differences between the two, especially including significant changes in the 1200–1300 cm⁻¹ (blue circle) region. (C) Spectral detail of the 1200–1300 cm⁻¹ region indicates that the biochemical changes likely occur around day 16.

Fig. 5

Analysis of stromal transformation around a tumor. (A) Spectra at tumor-neighboring dermal region at different depths and angles (R1–R7, R′1–R′7) are extracted. (B) Analysis of ROIs from the tumor and 7 peritumoral regions at both angles shows spectral differences. (C) CDM suggests a transition pattern of spectra from tumor to distal stromal regions at a few spectral peaks (highlighted by boxes in the picture), including a prominent peak 1200–1300 cm⁻¹ (red box). A sample from day 20 is used for the analysis.

Fig. 6

Chemical analysis of melanomastromal interactions. Regions of interest are extracted from tumors and from progressively increasing distance in stroma (R1–R7). Two duplicated samples from day 20, 24 and 28 are used. (A) The spectral region of 1200–1300 cm⁻¹, corresponding to collagen specific peaks, is presented to show the magnitude of changes. (B) CDM is used to visualize the differences among spectra. Regions of transition are highlighted in squares, including the collagen region (red square).