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ORIGINAL RESEARCH

Thermal Stability of M1 Sarcoma in Rat Tissue: A Differential Scanning Calorimetry Study
Manana Ramishvili1,ID, Maya Gorgoshidze2,ID, Revaz Monaselidze2, Jamlet Monaselidze2,ID
Received: 23 Jan 2026; Accepted: 25 Feb 2026; Available online: 2 Mar 2026
References
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ABSTRACT
Background: Clinical studies indicate that epithelial tissue is the source of over 90% of human cancers. Investigation of this phenomenon using the M1 rat sarcoma model and a new, rapid differential scanning calorimetry (DSC) method applied to milligram quantities of biopsied tissue enables further application of this approach for sarcoma diagnosis within 50-60 minutes. When used in combination with other diagnostic methods, this technique may facilitate timely diagnosis and improve treatment outcomes.

Objectives: (i) To evaluate the contribution of collagen – the major structural protein – and collagen fibrils to tissue thermal stability using a rat M1 sarcoma model based on DSC measurements; (ii) To determine the degree of disease progression based on differences in the thermal stability of postoperative M1 sarcoma tissue at 10, 20, and 30 days after resection compared with healthy tissue obtained from areas distant from the sarcoma.

Methods: In each experiment, tissue from three albino female rats was used (nine animals in total). Tumor tissue excised 10, 20, and 34 days after transplantation, together with healthy tissue obtained from sites distant from the tumor, was analyzed at 10-15 mg of fresh tissue using a highly sensitive differential scanning calorimeter (DSC). The instrument, designed for the study of complex biological systems, allows measurement of tissue denaturation with an accuracy of ±0.1 °C. This approach enables precise determination of temperature-induced structural transitions in biomacromolecules and their complexes.

Results: Differential scanning calorimetry (DSC) was performed on fresh postoperative rat tissues collected 10, 20, and 34 days after M1 sarcoma transplantation. Tumor volumes at these time points were 1.0±0.25 cm³, 10.05±2.4 cm³, and 37.5±4.2 cm³, respectively. Tissue samples were obtained from dense regions of M1 sarcoma and from healthy skin at sites distant from the tumor. In addition, healthy tissue, sarcoma tissue, and collagen fibrils isolated from healthy tissue were analyzed at different water contents.

Conclusions: DSC measurements showed that: The thermal stability of M1 sarcoma connective tissue was reduced by 2-3°C relative to normal tissue at early stages of the disease, increasing to approximately 5°C with further tumor growth. These findings suggest that such data may be used for tumor diagnosis at different stages of sarcoma progression, provided that sufficient experimental data are accumulated. Based on DSC measurements of native collagen fibrils isolated from healthy tissue, whole fresh healthy rat tissue, and fresh M1 sarcoma tissue at varying water contents, it is concluded that the reduced thermal stability of the connective tissue in M1 sarcoma compared with healthy tissue is associated with collagen.

Keywords: Collagen; extracellular matrix; differential scanning calorimetry (DSC); sarcoma M1; tissue dentauration.


DOI: 10.52340/GBMN.2026.01.01.154
BACKGROUND

Solid tumors constitute the majority of cancers and are predominantly associated with epithelial malignancies (carcinomas), which originate from epithelial tissues such as the skin, mammary gland, colon, and prostate. Clinical observations indicate that epithelial tissue is the source of over 90% of human cancers, most of which develop at sites of chronic inflammation and/or within senescent epithelium.1,2 This phenomenon is explained by the accumulation of genetic and epigenetic alterations in aging organisms, together with the chronic low-grade inflammatory state characteristic of senescent epithelium, which creates a pro-tumorigenic microenvironment resembling the tumor microenvironment (TME).

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The TME and its stromal components, including the extracellular matrix (ECM), play a critical role in tumor initiation, metastasis, recurrence, and drug resistance in epithelial cancers.3,4 The main structural components of the stromal matrix include type I collagen,5 elastin,6 fibrillin, and fibronectin (Fn).7 The latter is a plasma and cellular glycoprotein, fibronectin (cFn). This important molecule, which possesses diverse adhesive properties, exerts specific effects through interactions with cellular integrins. As a result, it undergoes conformational unfolding and effectively binds to collagen and the proteins mentioned above, forming tightly packed fibrils. One of the fibronectin modules, namely FN1, plays a key role in cancer. It consists of 12 FN1 repeats and is primarily localized within the 70 kDa fibronectin fragment. FN1, together with its binding partners and stromal and tumor cells, is actively involved in all stages of tumor initiation and progression.5,8,9

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In cancer, the expression and organization of FN1 change primarily through interactions with cellular integrins and the activation of cancer-associated fibroblasts (CAFs). As a result, FN1 levels increase, and its interactions with collagen, elastin, fibrillin, and fibulin fibrils are enhanced. Furthermore, these complexes undergo covalent cross-linking mediated by lysyl oxidase (LOX), leading to gradual thickening and increased rigidity of the fibrils and, consequently, of the tissue. This increased stiffness propagates toward regions of cancer cell invasion.4,8,9 Simultaneously, growth factors and matrix metalloproteinases (MMPs) secreted by stromal and tumor cells degrade the ECM and basement membrane, thereby facilitating tumor cell migration through lymphatic vessels and dissemination to distant organs, resulting in metastatic foci.

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Differential scanning calorimetry (DSC) is a powerful method for directly determining the energy required for temperature-induced protein unfolding, enabling assessment of the degree of nativeness and structural organization of biopolymers in solutions10 and in complex biological systems such as cells,11 blood plasma,11-15 and tissues.16-18 this study aims to demonstrate that the active collagen constitutes the main structural component of M1 sarcoma tissue and that a decrease in its thermal stability by 3–5°C relative to normal tissue, depending on disease stage, may serve as a diagnostic criterion for biopsied tissue from other sarcomas, including human sarcomas, provided that sufficient experimental data are available.

METHODS

All experiments were conducted in accordance with international ethical guidelines for medical and biological research involving animals (CIOMS, Geneva, 1985). Female mongrel rats bearing subcutaneously implanted M1 sarcoma were used in this study. Sarcoma transplantation and tumor volume determination were performed using a well-known methodology.19 Tumors were excised 10-, 20-, and 34-days post-transplantation to investigate their biophysical properties, with particular emphasis on the thermal stability of their major components during tumor development. Three rats were analyzed at each time point, for a total of nine animals. Over five years (2000–2005), three series of experiments were conducted.

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Differential scanning calorimetry (DSC) measurements were performed using a highly sensitive calorimeter specifically designed for the analysis of complex biological systems.20 The measuring cell volume was 30 µL, and the heating rate was set to 1°C/min (adjustable within the range of 0.1–2°C/min). The calorimeter sensitivity was 5 × 10⁻⁷ W, and the temperature measurement range was 20–140°C.

RESULTS

Differential scanning calorimetry curves

The figures below depict study results obtained using differential scanning calorimetry (DSC) to measure tissue obtained from albino female rats after transplantation of M1 sarcoma. (Fig.1 and Fig.2)


FIGURE 1. Heat absorption curves as a function of temperature (dQ/dT, Jg-1K-1) for rat sarcoma M1 tissue. The tissue mass ranged from 10.5 to 14.5 mg. The dQ/dT values were recalculated on a per-gram-of-dry-mass basis. Tissue samples were collected from healthy rats and from rats 10, 20, and 34 days after transplantation of M1 sarcoma
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FIGURE 2. Heat absorption curves as a function of temperature (dQ/dT, Jg⁻¹K⁻¹) for collagen fibers from healthy tissue and rat M1 sarcoma tissue at different water contents. Solid line – native-type fibrils
Explanations: Dash-dot line – healthy tissue. Dotted line – M1 sarcoma tissue. Inset – dependence of the denaturation temperature (corresponding to the maxima of the heat absorption curves) on water content. Black squares indicate native-type fibrils, white squares indicate M1 sarcoma tissue, and the black triangle indicates healthy tissue.
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DISCUSSION

In Figure 1, the DSC curves show that healthy tissue and M1 sarcoma stiff tissue collected 10 days after transplantation denature within a narrow temperature range (ΔTd=4±1°C). However, the denaturation temperature (Td) of M1 sarcoma tissue was 2-3°C lower than that of normal tissue (60.5±1°C). On days 20 and 34 after transplantation, the denaturation temperature further decreased to 58.5±1°C.

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In addition, tissues excised on days 20 and 34 exhibited two clearly defined shoulders on the DSC curves in the temperature ranges of 45-55°C and 67-71°C. According to published data,21,22 these features most likely correspond to the denaturation of collagen molecules and their native fibrils (Fig.2) and to the erythrocytic mass of whole blood, respectively.13,21

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The melting enthalpy (ΔHd), defined as the energy required to denaturate biomacromolecules and their supramolecular structures, is reduced by 20-25% compared with normal tissue and amounts to 45±5 Jg⁻¹ of dry protein weight. We attribute this decrease to cancer-associated proteins that lack well-defined secondary and tertiary structures, unlike compact globular proteins.23 This interpretation is further supported by a significant broadening of the melting range (ΔTd) on days 20 and 34. Together, these thermodynamic parameters directly indicate progressive destruction of the M1 sarcoma tissue structure.

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As shown in Figure 2, a full removal of the free water and a partial removal of the bound water in the samples leads to a rise in the transition temperatures (Td - temperatures corresponding to the maxima of heat absorption peaks) for M1 sarcoma tissue, healthy tissue, and collagen fibrils by approximately 45-50°C (see insert in Fig.2). This behavior indicates that collagen fibers are the predominant structural component of M1 sarcoma tissue, as such a pronounced increase in Td with decreasing water content is observed only in collagen molecules, due to their orientation in a single direction, which forms ordered structures similar to biological crystals.20,21 Weak, diffuse endothermic peaks observed at 50-70°C and 70-90°C correspond to the denaturation of the remaining proteins and protein complexes in the tissue.

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Cellular fibronectin (cFn), which, unlike plasma fibronectin, has a fibrillar structure and is present in tissues at concentrations approximately ten times lower than in plasma (30–40 μg/mL),24 significantly contributes to the rigidity of the extracellular matrix (ECM) mainly in concert with collagen fibrils. This integrated structural network plays a key role in tumor growth, initiation, and metastatic progression.12

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Based on these findings, we conclude that the observed 3-5°C decrease in the thermodynamic stability of M1 sarcoma tissue relative to normal (healthy) tissue is directly related to ECM collagen, and that this parameter can be used for the diagnosis and monitoring of treatment in various sarcomas.

CONCLUSIONS

The 3-5°C reduction in the denaturation temperature of M1 sarcoma tissue compared with healthy tissue, as determined by milligram-scale DSC measurements, suggests strong potential for this approach as a rapid, reliable DSC-based test for detecting various sarcomas, including human sarcomas.

AUTHOR AFFILIATION

1 Department of Topographic Anatomy & Operative Surgery, Tbilisi State Medical University, Tbilisi, Georgia

2 E. Andronikashvili Institute of Physics, I. Javakhishvili Tbilisi State University, Tbilisi, Georgia

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