2025 Carolina Palacios-Montoya, Andrés Milán, Valeria Hernández, Karen Rodríguez, Marco Giraldo, Robson Carvalho, Geysson Fernández, Juan C. Calderón. Muscle atrophy in lung cancer cachexia: towards a living single muscle fiber approach 1 Background Data version: Globocan 2022 (version 1.1) - 08.02.2024 cc is a multifactorial syndrome characterized by involuntary weight loss with irreversible consumption of muscle mass and adipose tissue, leading to a reduction in patients' quality of life, vulnerability to infections and limited treatment. In these maps I show the incidence and mortality of cc, which affects 50-80% of patients with advanced cancer and alone leads to a mortality of 20-30% of patients. there is a need to study this syndrome, and we have some gaps that we need to address, one of them is that we do not know what happens in the isolated muscle fiber nor do we know how the calcium flow inside the muscle fiber is altered. we do not know the differences in the effect between sexes and we do not have a clear model for studying cc. This model will increase the resolution to compare muscle wasting in females vs males, uncover critical mechanisms underlying cachexia and guide the specific search for novel therapeutic targets. Hypothesis It is possible to develop a living single-fiber experimental murine model to study the lung cancer cachexia-induced muscle atrophy. Objective To develop a living, single-muscle fiber murine model of lung cancer cachexia. Hypothesis and objective we have the approval of the ethics committee LLC1 Passage 4 or 5 Methods Ethical approval: minutes 138 of February 9th, 2021, UdeA, Colombia 1x106 cells - PBS 200 µL 1. Culture 2. Inoculation 3. Following Measurements 2. Tibia length 3. Blood EDL, soleus, PL, FDB, PT, gastrocnemius 5. Physiological experiments 4. Weights Post-euthanasia Regarding the muscles, some were frozen for future experiments, others were subjected to enzymatic dissociation with collagenase type iv, once the fibers were dissociated they were photographed for diameter measurement and loaded with magfluo 4 am for the capture of calcium transients. Results Measurements at 4th week 2. Weights of organs 3. Fiber diameters p= 0.02 p= 0.345 p= 0.63 FEMALES Figure 1: Weight measurements are shown on the left Y-axis, and calf and thigh circumference are displayed on the right Y-axis, comparing the tumor-bearing and control conditions for both female (A) and male (B) mice. p= 0.034 p= 0.279 p= 0.285 MALES B A Weight and circumferences at 4th week now, I present you initially these figures that represent what happened in the fourth week with the 3 main measurements we were interested in. in panel A I present the graph that shows the weight at week 4, and the thigh and calf perimeters of controls vs. tumors, we see how a significant p is observed in the change of weight, being a greater weight for the tb, this due to the size and weight of the tumor. and we see how there is a tendency in reduction of the thigh and calf perimeters. in panel B and c is the sex difference of the same parameters. let's observe how males approach statistical significance in thigh and calf perimeters, inconclusion we have a promising results. A p= 0.143 B p= 0.006 p= 0.072 C Control initial Control final TB initial TB final Figure 2: Initial vs. final weight without tumor. n = 4 for controls, and n = 5 for tumor-bearing measurements. Weight without tumor FEMALES MALES We then decided to use for comparison, the weight of the last day after tumor removal, and contrast it with the initial weight. in panel A we see the general model for both sexes, we observe that the p is significant for the general model, here the controls gain weight and the tumors neither gain nor lose weight but when we see the comparison separating by sex in panel b and c there is a clear loss of weight for the TB females comparing the last day vs the first day and the males do not seem to have such a significant reduction. We decided to calculate the delta of the initial vs final weights, obtaining a significant p. look how controls gain near to 2 gr and TB lose near to 2 gr, I mean the final delta is about 4 gr. this data confirm the model of cachexia and in spite low sample size, the change was so big that we see a significant p linear repeated measures model Figure 3: Organs weights normalized by tibia length. Scale bar: 1 mm. n = 9 (4 controls and 5 tumor-bearing mice for each organ and fat type). FDB: Flexor Digitorum Brevis; EDL: Extensor Digitorum Longus; SOL: Soleus; PL, Peroneus Longus; PT: plantaris; GAS: Gastrocnemius. p= 0.037 Organs and tissue weight A B C p= 0.1 to 0.4 p= 0.318 initially I present this graph, in the upper part of panel A, the organs in pink controls vs. in blue TB, and in the lower part on the Y axis the weight value in grams normalized by the length of the tibia of each mouse, I want to highlight the size of the spleen, which is clearly larger with a significant p in TB vs. controls, and compare it with the images at the top. In panel B we have the fats also normalized and in spite of not having a significant p we observe a reduction in all the TB fats. p= 0.001 p= 0.000 p= 0.02 p= 0.5 C A B Living isolated muscle fibers Figure 4: Representative images of FDB Muscle Fibers from a control mouse (A) and a Tumor-Bearing (TB) mouse (B). FDB n=83 control, n=134 TB; EDL n=14 control, n=18 TB; PL n=14 control, n=47 TB; SOL n=5 control, n=2 TB. I want to visually show you the difference between a mouse control fiber on the left and a cachectic mouse fiber on the right. You can see that the cell is intact with its membrane intact and retains the expected transverse striation pattern. Figure 5: Fiber diameters for female and male. FDB n=43 female, 40 male control, n=42 female, 92 male TB; EDL n=4 female, 10 male control, n=7 female, 11 male TB; PL n=9 female, 5 male control, n=19 female, 28 male TB; SOL n=2 female, 3 male control, n=2 female TB. p= 0.014 p= 0.732 p= 0.667 p= 0.24 p= 0.001 p= 0.001 p= 0.002 Living isolated muscle fibers A B FEMALES MALES now analyzing the comparison between sexes, it is evident how the females in panel b apparently only atrophied fdb with statistical significance, the other fibers show tendency, we will be able to conclude this part once the N is increased, in contrast, the males atrophied the fibers of fdb edl and pl. Conclusions 1. We developed a living single-fiber experimental murine model to study the lung cancer cachexia-induced muscle atrophy in males and females. 2. Despite females showed bigger tumors and more wasting, their single muscle fibers were spared. 3.These results open the possibility for conducting molecular and physiological studies at the living single-muscle fiber level. 12 N n 13 image1.png image2.png image3.png image4.png image5.png image6.png image7.png image8.png image9.png image10.png image11.png image17.png image18.png image19.png image20.png image21.png image22.png image23.png image24.png image12.png image13.png image14.png image15.png image16.png image28.png image29.png image30.png image31.png image32.png image33.png image25.png image26.png image27.png image34.png image35.png image36.png image37.png image38.png image39.png image40.png image41.png image42.png image43.png image44.png image45.png image49.png media1.mp4 image46.png image47.jpg image48.jpg image50.png image51.png image52.png image53.png image54.png image55.png image56.png